U.S. patent application number 15/845572 was filed with the patent office on 2018-05-03 for degradable clostridial toxins.
The applicant listed for this patent is Allergan, Inc.. Invention is credited to Kei Roger Aoki, Ester Fernandez-Salas, Joseph Francis, Sanjiv Ghanshani, Marcella A. Gilmore, Lance E. Steward.
Application Number | 20180119126 15/845572 |
Document ID | / |
Family ID | 44484018 |
Filed Date | 2018-05-03 |
United States Patent
Application |
20180119126 |
Kind Code |
A1 |
Steward; Lance E. ; et
al. |
May 3, 2018 |
DEGRADABLE CLOSTRIDIAL TOXINS
Abstract
The specification discloses Clostridial toxins or Clostridial
toxin chimeras comprising an inactivation cleavage site,
polynucleotide molecules encoding such toxins or chimeras,
compositions comprising such toxins or chimeras, and method of
producing such toxins or chimeras.
Inventors: |
Steward; Lance E.; (Irvine,
CA) ; Ghanshani; Sanjiv; (Irvine, CA) ;
Fernandez-Salas; Ester; (Ann Arbor, MI) ; Gilmore;
Marcella A.; (Santa Ana, CA) ; Francis; Joseph;
(Aliso Viejo, CA) ; Aoki; Kei Roger; (Coto de
Caza, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc. |
Irvine |
CA |
US |
|
|
Family ID: |
44484018 |
Appl. No.: |
15/845572 |
Filed: |
December 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15082635 |
Mar 28, 2016 |
9850476 |
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15845572 |
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14088022 |
Nov 22, 2013 |
9297003 |
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15082635 |
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13846364 |
Mar 18, 2013 |
8841111 |
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14088022 |
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13112844 |
May 20, 2011 |
8512992 |
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13846364 |
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61346578 |
May 20, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 5/00 20180101; A61P
17/00 20180101; A61P 27/02 20180101; A61P 35/00 20180101; C07K
14/33 20130101; C12Y 304/24069 20130101; A61P 9/00 20180101; A61P
25/06 20180101; A61P 25/00 20180101; C12N 9/52 20130101; A61P 27/16
20180101; A61K 38/4893 20130101; A61P 25/04 20180101; A61P 21/00
20180101; A61P 25/02 20180101 |
International
Class: |
C12N 9/52 20060101
C12N009/52; A61K 38/48 20060101 A61K038/48; C07K 14/33 20060101
C07K014/33 |
Claims
1. A Clostridial toxin comprising at least one inactivation
cleavage site located within an inactivation cleavage site region,
wherein the inactivation cleavage site region is located in the
translocation domain and/or the H.sub.CN binding sub domain,
wherein the at least one inactivation cleavage site comprises a
dual Thrombin-Thrombin site, a Factor Xa site, a dual Factor
Xa-Thrombin site, and/or a MMP-9 site, and wherein the inactivation
cleavage site region comprises amino acids 871-895 of SEQ ID NO:
1.
2. The Clostridial toxin of claim 1, wherein the Clostridial toxin
enzymatic domain comprises a BoNT/A enzymatic domain.
3. The Clostridial toxin of claim 1, comprising an amino acid
sequence selected from the group consisting of SEQ ID: 531, SEQ ID
NO: 533, SEQ ID NO: 535 and SEQ ID NO: 537.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/082,635, filed Mar. 28, 2016, now U.S. Pat. No. 9,850,476,
which is a continuation of U.S. application Ser. No. 14/088,022,
filed Nov. 22, 2013, now U.S. Pat. No. 9,297,003, which is a
divisional and claims priority pursuant to 35 U.S.C. .sctn. 120 to
U.S. patent application Ser. No. 13/846,364, filed Mar. 18, 2013,
now U.S. Pat. No. 8,841,111, which claims priority to U.S. patent
application Ser. No. 13/112,844, filed May 20, 2011, now U.S. Pat.
No. 8,512,992, which claims priority pursuant to 35 U.S.C. .sctn.
119(e) to U.S. Provisional Patent Application Ser. No. 61/346,578,
filed on May 20, 2010, all incorporated entirely by reference.
[0002] The ability of Clostridial toxins, such as, e.g., Botulinum
neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,
BoNT/F and BoNT/G, and Tetanus neurotoxin (TeNT), to inhibit
neuronal transmission are being exploited in a wide variety of
therapeutic and cosmetic applications, see e.g., William J. Lipham,
COSMETIC AND CLINICAL APPLICATIONS OF BOTULINUM TOXIN (Slack, Inc.,
2004). Clostridial toxins commercially available as pharmaceutical
compositions include, BoNT/A preparations, such as, e.g.,
BOTOX.RTM. (Allergan, Inc., Irvine, Calif.),
DYSPORT.RTM./RELOXIN.RTM., (Beaufour Ipsen, Porton Down, England),
NEURONOX.RTM. (Medy-Tox, Inc., Ochang-myeon, South Korea) BTX-A
(Lanzhou Institute Biological Products, China) and XEOMIN.RTM.
(Merz Pharmaceuticals, GmbH., Frankfurt, Germany); and BoNT/B
preparations, such as, e.g., MYOBLOC.TM./NEUROBLOC.TM. (Elan
Pharmaceuticals, San Francisco, Calif.). As an example, BOTOX.RTM.
is currently approved in one or more countries for the following
indications: achalasia, adult spasticity, anal fissure, back pain,
blepharospasm, bruxism, cervical dystonia, essential tremor,
glabellar lines or hyperkinetic facial lines, headache, hemifacial
spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral
palsy, multiple sclerosis, myoclonic disorders, nasal labial lines,
spasmodic dysphonia, strabismus and VII nerve disorder.
[0003] A Clostridial toxin treatment inhibits neurotransmitter
release by disrupting the exocytotic process used to secrete the
neurotransmitter into the synaptic cleft. There is a great desire
by the pharmaceutical industry to expand the use of Clostridial
toxin therapies beyond its current myo-relaxant applications to
treat sensory nerve-based ailments, such as, e.g., various kinds of
chronic pain, neurogenic inflammation and urogenital disorders, as
well as other disorders, such as, e.g., pancreatitis. One approach
that is currently being exploited to expand Clostridial toxin-based
therapies involves modifying a Clostridial toxin so that the
modified toxin has an altered cell targeting capability for a
non-Clostridial toxin target cell. This re-targeted capability is
achieved by replacing a naturally-occurring targeting domain of a
Clostridial toxin with a targeting domain showing a preferential
binding activity for a non-Clostridial toxin receptor present in a
non-Clostridial toxin target cell. Such modifications to a
targeting domain result in a Clostridial toxin chimeric called a
Targeted Vesicular Exocytosis Modulating Protein (TVEMP) that is
able to selectively bind to a non-Clostridial toxin receptor
(target receptor) present on a non-Clostridial toxin target cell
(re-targeted). A Clostridial toxin chimeric with a targeting
activity for a non-Clostridial toxin target cell can bind to a
receptor present on the non-Clostridial toxin target cell,
translocate into the cytoplasm, and exert its proteolytic effect on
the SNARE complex of the non-Clostridial toxin target cell.
[0004] Clostridial toxin and Clostridial toxin chimeric therapies
are successfully used for many indications. Generally,
administration of a Clostridial toxin or Clostridial toxin chimeric
is well tolerated. However, administration in some applications can
be challenging because of the larger doses required to achieve a
beneficial effect. Larger doses can increase the likelihood that
the toxin or Clostridial toxin chimeric may move through the
interstitial fluids and the circulatory systems, such as, e.g., the
cardiovascular system and the lymphatic system, of the body,
resulting in the undesirable dispersal of the toxin or Clostridial
toxin chimeric to areas not targeted for treatment. Such dispersal
can lead to undesirable side effects, such as, e.g., inhibition of
neurotransmitter release in neurons not targeted for toxin
treatment or paralysis of a muscle not targeted for treatment. For
example, a patient administered a therapeutically effective amount
of a BoNT/A treatment into the neck muscles for torticollis may
develop dysphagia because of dispersal of the toxin into the
oropharynx. Thus, there remains a need for improved Clostridial
toxins and/or Clostridial toxin chimeras that are effective at the
site of treatment, but have negligible to minimal effects in areas
not targeted for toxin treatment.
[0005] The growing clinical, therapeutic, and cosmetic use of
Clostridial toxins and Clostridial toxin chimeras in therapies
requiring larger doses necessitates the pharmaceutical industry to
develop modified Clostridial toxins and Clostridial toxin chimeras
that are effective at the target site of application, but reduce or
prevent the possible side-effects associated with the dispersal of
the toxins to an unwanted location. The present specification
provides novel modified Clostridial toxins and Clostridial toxin
chimeras that reduce or prevent unwanted side-effects associated
with toxin dispersal into non-targeted areas. These and related
advantages are useful for various clinical, therapeutic and
cosmetic applications, such as, e.g., the treatment of
neuromuscular disorders, neuropathic disorders, eye disorders,
pain, muscle injuries, headache, cardiovascular diseases,
neuropsychiatric disorders, endocrine disorders, cancers, otic
disorders and hyperkinetic facial lines, as well as, other
disorders where a Clostridial toxin or a Clostridial toxin chimeric
administration to a mammal can produce a beneficial effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGS. 1A and 1B show a schematic of the current paradigm of
neurotransmitter release and Clostridial toxin intoxication in a
central and peripheral neuron. FIG. 1A shows a schematic for the
neurotransmitter release mechanism of a central and peripheral
neuron. The release process can be described as comprising two
steps: 1) vesicle docking, where the vesicle-bound SNARE protein of
a vesicle containing neurotransmitter molecules associates with the
membrane-bound SNARE proteins located at the plasma membrane; and
2) neurotransmitter release, where the vesicle fuses with the
plasma membrane and the neurotransmitter molecules are exocytosed.
FIG. 1B shows a schematic of the intoxication mechanism for tetanus
and botulinum toxin activity in a central and peripheral neuron.
This intoxication process can be described as comprising four
steps: 1) receptor binding, where a Clostridial toxin binds to a
Clostridial receptor system and initiates the intoxication process;
2) complex internalization, where after toxin binding, a vesicle
containing the toxin/receptor system complex is endocytosed into
the cell; 3) light chain translocation, where multiple events are
thought to occur, including, e.g., changes in the internal pH of
the vesicle, formation of a channel pore comprising the H.sub.N
domain of the Clostridial toxin heavy chain, separation of the
Clostridial toxin light chain from the heavy chain, and release of
the active light chain and 4) enzymatic target modification, where
the activate light chain of Clostridial toxin proteolytically
cleaves its target SNARE substrate, such as, e.g., SNAP-25, VAMP or
Syntaxin, thereby preventing vesicle docking and neurotransmitter
release.
[0007] FIG. 2 shows the domain organization of naturally-occurring
Clostridial toxins. The single-chain form depicts the amino to
carboxyl linear organization comprising an enzymatic domain, a
translocation domain, and a binding domain. The di-chain loop
region located between the translocation and enzymatic domains is
depicted by the double SS bracket. This region comprises an
endogenous di-chain loop protease cleavage site that upon
proteolytic cleavage with a naturally-occurring protease, such as,
e.g., an endogenous Clostridial toxin protease or a
naturally-occurring protease produced in the environment, converts
the single-chain form of the toxin into the di-chain form. Above
the single-chain form, the H.sub.CC region of the Clostridial toxin
binding domain is depicted. This region comprises the n-trefoil
domain which comprises in an amino to carboxyl linear organization
an .alpha.-fold, a .beta.4/.beta.5 hairpin turn, a .beta.-fold, a
.beta.8/.beta.9 hairpin turn, and a .gamma.-fold.
[0008] FIGS. 3A and 3B show Clostridial toxins or Clostridial toxin
chimeras with a binding domain located at the amino terminus of the
toxin. FIG. 3A depicts the single-chain polypeptide form of a toxin
or chimera with an amino to carboxyl linear organization comprising
a binding element, a translocation element, a di-chain loop region
comprising an exogenous protease cleavage site (P), and a
therapeutic element. Upon proteolytic cleavage with a P protease,
the single-chain form of the toxin or chimera is converted to the
di-chain form. FIG. 3B depicts the single polypeptide form of a
toxin or chimera with an amino to carboxyl linear organization
comprising a binding element, a therapeutic element, a di-chain
loop region comprising an exogenous protease cleavage site (P), and
a translocation element. Upon proteolytic cleavage with a P
protease, the single-chain form of the toxin or chimera is
converted to the di-chain form.
[0009] FIGS. 4A, 4B, 4C and 4D show Clostridial toxins or
Clostridial toxin chimeras with a binding domain located at the
amino terminus of the toxin. FIG. 4A depicts the single polypeptide
form of a toxin or chimera with an amino to carboxyl linear
organization comprising a therapeutic element, a di-chain loop
region comprising an exogenous protease cleavage site (P), a
binding element, and a translocation element. Upon proteolytic
cleavage with a P protease, the single-chain form of the toxin or
chimera is converted to the di-chain form. FIG. 4B depicts the
single polypeptide form of a toxin or chimera with an amino to
carboxyl linear organization comprising a translocation element, a
di-chain loop region comprising an exogenous protease cleavage site
(P), a binding element, and a therapeutic element. Upon proteolytic
cleavage with a P protease, the single-chain form of the toxin or
chimera is converted to the di-chain form. FIG. 4C depicts the
single polypeptide form of a toxin or chimera with an amino to
carboxyl linear organization comprising a therapeutic element, a
binding element, a di-chain loop region comprising an exogenous
protease cleavage site (P), and a translocation element. Upon
proteolytic cleavage with a P protease, the single-chain form of
the toxin or chimera is converted to the di-chain form. FIG. 4D
depicts the single polypeptide form of a toxin or chimera with an
amino to carboxyl linear organization comprising a translocation
element, a binding element, a di-chain loop region comprising an
exogenous protease cleavage site (P), and a therapeutic element.
Upon proteolytic cleavage with a P protease, the single-chain form
of the toxin or chimera is converted to the di-chain form.
[0010] FIGS. 5A and 5B show Clostridial toxins or Clostridial toxin
chimeras with a binding domain located at the amino terminus of the
toxin. FIG. 5A depicts the single polypeptide form of a toxin or
chimera with an amino to carboxyl linear organization comprising a
therapeutic element, a di-chain loop region comprising an exogenous
protease cleavage site (P), a translocation element, and a binding
element. Upon proteolytic cleavage with a P protease, the
single-chain form of the toxin is converted to the di-chain form.
FIG. 5B depicts the single polypeptide form of a toxin or chimera
with an amino to carboxyl linear organization comprising a
translocation element, a di-chain loop region comprising an
exogenous protease cleavage site (P), a therapeutic element, and a
binding element. Upon proteolytic cleavage with a P protease, the
single-chain form of the toxin or chimera is converted to the
di-chain form.
DETAILED DESCRIPTION
[0011] The present specification discloses modified Clostridial
toxins and modified Clostridial toxin chimeras that can be rapidly
inactivated from an unwanted location or locations by exploiting
the presence of proteases present in interstitial fluids and
circulatory systems, such as, e.g., the cardiovascular system and
the lymphatic system. This is because the modified Clostridial
toxins and modified Clostridial toxin chimeras disclosed in the
present specification comprise a protease cleavage site for a
protease present in an interstitial fluid and/or a circulatory
system. The presence of such a protease cleavage site makes the
modified Clostridial toxin or modified Clostridial toxin chimeric
susceptible to proteolytic cleavage by its cognate protease, which
renders such modified toxins inactive. For example, in situations
where a Clostridial toxin or Clostridial toxin chimeric modified to
comprise a cleavage site for an extracellular matrix protease has
diffused into the interstitial fluid, this modified toxin or
modified Clostridial toxin chimeric can be effectively cleaved by
the cognate extracellular matrix protease. As another example, in
situations where a Clostridial toxin or Clostridial toxin chimeric
modified to comprise a cleavage site for a blood protease has
diffused into the cardiovascular system, this modified toxin or
modified Clostridial toxin chimeric can be effectively cleaved by
the cognate blood protease. As yet another example, in situations
where a Clostridial toxin or Clostridial toxin chimeric modified to
comprise a cleavage site for a lymphatic protease has diffused into
the lymphatic system, this modified toxin or modified Clostridial
toxin chimeric can be effectively cleaved by the cognate lymphatic
protease. Thus utilizing a Clostridial toxin or Clostridial toxin
chimeric comprising a cleavage site(s) for proteases present the
interstitial fluid and/or circulatory system will lessen or remove
such Clostridial toxin or Clostridial toxin chimeric from an
unwanted location, thereby reducing or preventing the undesirable
side-effects associated with the diffusion of a Clostridial toxin
or Clostridial toxin chimeric to an unwanted location.
[0012] Thus, aspects of the present specification provide a
Clostridial toxin comprising an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain or the H.sub.CN binding subdomain. Such disclosed toxins can
comprise a Clostridial toxin enzymatic domain, a Clostridial toxin
translocation domain, a Clostridial toxin binding domain, a
di-chain loop region comprising an exogenous protease cleavage
site, and an inactivation cleavage site located within an
inactivation cleavage site region. Non-limiting examples of
inactivation cleavage sites include Thrombin cleavage sites,
Plasmin cleavage sites, Coagulation Factor VIIa cleavage sites,
Coagulation Factor IXa cleavage sites, Coagulation Factor Xa
cleavage sites, Coagulation Factor XIa cleavage sites, Coagulation
Factor XIIa cleavage sites, plasma kallikrein cleavage sites,
protease-activated G protein-coupled receptor-1 (PAR1) cleavage
sites, PAR2 cleavage sites, PAR3 cleavage sites, PAR4 cleavage
sites, Matrix Metalloproteinase-2 (MMP-2) cleavage sites, Matrix
Metalloproteinase-9 (MMP-9) cleavage sites, Furin cleavage sites,
urokinase-type Plasminogen activator (uPA) cleavage sites,
tissue-type Plasminogen activator (tPA) cleavage sites,
Tryptase-.epsilon. cleavage sites, Mouse mast cell protease-7
(mMCP-7) cleavage sites, endothelin-converting enzyme-1 (ECE-1)
cleavage sites, Kell blood group cleavage sites, DPPIV cleavage
sites, ADAM metallopeptidase with thrombospondin type 1 motif-13
(ADAMTS13) cleavage sites, and Cathepsin L cleavage sites. The
addition of the inactivation cleavage site increases the safety
margin of the Clostridial toxin or Clostridial toxin chimeric
relative to the same or similar Clostridial toxin or Clostridial
toxin chimeric, but without the additional inactivation cleavage
site.
[0013] Other aspects of the present specification provide a
Clostridial toxin chimeric comprising a Clostridial toxin enzymatic
domain, a Clostridial toxin translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain or the H.sub.CN binding subdomain. Such disclosed toxins can
comprise a Clostridial toxin enzymatic domain, a Clostridial toxin
translocation domain, a non-Clostridial toxin binding domain, a
di-chain loop region comprising an exogenous protease cleavage
site, and an inactivation cleavage site located within an
inactivation cleavage site region. Non-limiting examples of
inactivation cleavage sites include Thrombin cleavage sites,
Plasmin cleavage sites, Coagulation Factor VIIa cleavage sites,
Coagulation Factor IXa cleavage sites, Coagulation Factor Xa
cleavage sites, Coagulation Factor XIa cleavage sites, Coagulation
Factor XIIa cleavage sites, plasma kallikrein cleavage sites,
protease-activated G protein-coupled receptor-1 (PAR1) cleavage
sites, PAR 2 cleavage sites, PAR3 cleavage sites, PAR4 cleavage
sites, Matrix Metalloproteinase-2 (MMP-2) cleavage sites, Matrix
Metalloproteinase-9 (MMP-9) cleavage sites, Furin cleavage sites,
urokinase-type Plasminogen activator (uPA) cleavage sites,
tissue-type Plasminogen activator (tPA) cleavage sites,
Tryptase-.epsilon. cleavage sites, Mouse mast cell protease-7
(mMCP-7) cleavage sites, endothelin-converting enzyme-1 (ECE-1)
cleavage sites, Kell blood group cleavage sites, DPPIV cleavage
sites, ADAM metallopeptidase with thrombospondin type 1 motif-13
(ADAMTS13) cleavage sites, and Cathepsin L cleavage sites. The
addition of the inactivation cleavage site increases the safety
margin of the Clostridial toxin or Clostridial toxin chimeric
relative to the same or similar Clostridial toxin or Clostridial
toxin chimeric, but without the additional inactivation cleavage
site.
[0014] Other aspects of the present specification provide
polynucleotide molecules encoding a Clostridial toxin or a
Clostridial toxin chimeric disclosed in the present specification.
A polynucleotide molecule encoding such a Clostridial toxin or a
Clostridial toxin chimeric can further comprise an expression
vector.
[0015] Other aspects of the present specification provide a
composition comprising a Clostridial toxin or a Clostridial toxin
chimeric disclosed in the present specification. A composition
comprising such a Clostridial toxin or a Clostridial toxin chimeric
can be a pharmaceutical composition. Such a pharmaceutical
composition can comprise, in addition to a modified Clostridial
toxin disclosed in the present specification a pharmaceutical
carrier, a pharmaceutical component, or both.
[0016] Other aspects of the present specification provide a method
of producing a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification, the method comprising the
step of expressing in a cell a polynucleotide molecule encoding a
Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification, wherein expression from the polynucleotide
molecule produces the encoded Clostridial toxin or Clostridial
toxin chimeric. In other aspects, the method comprises the steps of
introducing into a cell a polynucleotide molecule encoding a
Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification, and expressing the polynucleotide molecule,
wherein expression from the polynucleotide molecule produces the
encoded Clostridial toxin or Clostridial toxin chimeric.
[0017] Clostridia toxins produced by Clostridium botulinum,
Clostridium tetani, Clostridium baratii and Clostridium butyricum
are the most widely used in therapeutic and cosmetic treatments of
humans and other mammals. Strains of C. botulinum produce seven
antigenically-distinct types of Botulinum toxins (BoNTs), which
have been identified by investigating botulism outbreaks in man
(BoNT/A, /B, /E and /F), animals (BoNT/C1 and /D), or isolated from
soil (BoNT/G). BoNTs possess approximately 35% amino acid identity
with each other and share the same functional domain organization
and overall structural architecture. It is recognized by those of
skill in the art that within each type of Clostridial toxin there
can be subtypes which differ somewhat in their amino acid sequence,
and also in the nucleic acids encoding these proteins. For example,
there are presently five BoNT/A subtypes, BoNT/A1, BoNT/A2,
BoNT/A3, BoNT/A4, and BoNT/A5, with specific subtypes showing about
84% to 93% amino acid identity when compared to the BoNT/A subtype
of SEQ ID NO: 1. As another example, there are presently five
BoNT/B subtypes, BoNT/B1, BoNT/B2, BoNT/B3, BoNT/Bnp, and BoNT/Bbv,
with specific subtypes showing about 93% to 96% amino acid identity
when compared to the BoNT/B subtype of SEQ ID NO: 6. As yet another
example, there are presently three BoNT/E subtypes, BoNT/E1,
BoNT/E2, and BoNT/E3, with specific subtypes showing about 95% to
99% amino acid identity when compared to the BoNT/E subtype of SEQ
ID NO: 15. While all seven BoNT serotypes have similar structure
and pharmacological properties, each also displays heterogeneous
bacteriological characteristics. In contrast, tetanus toxin (TeNT)
is produced by a uniform group of C. tetani. Two other Clostridia
species, C. baratii and C. butyricum, produce toxins, BaNT and
BuNT, which are similar to BoNT/F and BoNT/E, respectively.
[0018] Clostridial toxins are each translated as a single chain
polypeptide of approximately 150 kDa that is subsequently cleaved
by proteolytic scission within a disulfide loop by a
naturally-occurring protease (FIG. 1). This cleavage occurs within
the discrete di-chain loop region created between two cysteine
residues that form a disulfide bridge. This posttranslational
processing yields a di-chain molecule comprising an approximately
50 kDa light chain (LC) and an approximately 100 kDa heavy chain
(HC) held together by the single disulfide bond and non-covalent
interactions between the two chains. The naturally-occurring
protease used to convert the single chain molecule into the
di-chain is currently not known. In some serotypes, such as, e.g.,
BoNT/A, the naturally-occurring protease is produced endogenously
by the bacteria serotype and cleavage occurs within the cell before
the toxin is released into the environment. However, in other
serotypes, such as, e.g., BoNT/E, the bacterial strain appears not
to produce an endogenous protease capable of converting the single
chain form of the toxin into the di-chain form. In these
situations, the toxin is released from the cell as a single-chain
toxin which is subsequently converted into the di-chain form by a
naturally-occurring protease found in the environment.
[0019] Each mature di-chain molecule comprises three functionally
distinct domains: 1) an enzymatic domain located in the LC that
includes a metalloprotease region containing a zinc-dependent
endopeptidase activity which specifically targets core components
of the neurotransmitter release apparatus; 2) a translocation
domain contained within the amino-terminal half of the HC (H.sub.N)
that facilitates release of the LC from intracellular vesicles into
the cytoplasm of the target cell; and 3) a binding domain found
within the carboxyl-terminal half of the HC (H.sub.C) that
determines the binding activity and binding specificity of the
toxin to the receptor complex located at the surface of the target
cell. D. B. Lacy and R. C. Stevens, Sequence Homology and
Structural Analysis of the Clostridial Neurotoxins, J. Mol. Biol.
291: 1091-1104 (1999). The H.sub.C domain comprises two distinct
structural features of roughly equal size, separated by an
.alpha.-helix, designated the H.sub.CN and H.sub.CC subdomains.
Table 1 gives approximate boundary regions for each domain and
subdomain found in exemplary Clostridial toxins.
TABLE-US-00001 TABLE 1 Clostridial Toxin Reference Sequences and
Regions SEQ ID Di-Chain H.sub.C Toxin NO: LC Loop H.sub.N H.sub.CN
.alpha.-Linker H.sub.CC BoNT/A 1 M1/P2-L429 C430-C454 I455-I873
I874-N1080 E1081-Q1091 S1092-L1296 BoNT/B 6 M1/P2-M436 C437-C446
I447-I860 L861-S1067 Q1068-Q1078 S1079-E1291 BoNT/C1 11 M1/P2-F436
C437-C453 R454-I868 N869-D1081 G1082-L1092 Q1093-E1291 BoNT/D 13
M1/T2-V436 C437-C450 I451-I864 N865-S1069 N1069-Q1079 I1080-E1276
BoNT/E 15 M1/P2-F411 C412-C426 I427-I847 K848-D1055 E1056-E1066
P1067-K1252 BoNT/F 18 M1/P2-F428 C429-C445 I446-I865 K866-D1075
K1076-E1086 P1087-E1274 BoNT/G 21 M1/P2-M435 C436-C450 I451-I865
S866-N1075 A1076-Q1086 S1087-E1297 TeNT 22 M1/P2-L438 C439-C467
I468-L881 K882-N1097 P1098-Y1108 L1109-D1315 BaNT 23 M1/P2-L420
C421-C435 I436-I857 I858-D1064 K1065-E1075 P1076-E1268 BuNT 24
M1/P2-F411 C412-C426 I427-I847 K848-D1055 E1056-E1066
P1067-K1251
[0020] The binding, translocation, and enzymatic activity of these
three functional domains are all necessary for toxicity. While all
details of this process are not yet precisely known, the overall
cellular intoxication mechanism whereby Clostridial toxins enter a
neuron and inhibit neurotransmitter release is similar, regardless
of serotype or subtype. Although the applicants have no wish to be
limited by the following description, the intoxication mechanism
can be described as comprising at least four steps: 1) receptor
binding, 2) complex internalization, 3) light chain translocation,
and 4) enzymatic target modification (FIG. 3). The process is
initiated when the H.sub.C domain of a Clostridial toxin binds to a
toxin-specific receptor system located on the plasma membrane
surface of a target cell. The binding specificity of a receptor
complex is thought to be achieved, in part, by specific
combinations of gangliosides and protein receptors that appear to
distinctly comprise each Clostridial toxin receptor complex. Once
bound, the toxin/receptor complexes are internalized by endocytosis
and the internalized vesicles are sorted to specific intracellular
routes. The translocation step appears to be triggered by the
acidification of the vesicle compartment. This process seems to
initiate two important pH-dependent structural rearrangements that
increase hydrophobicity and promote formation di-chain form of the
toxin. Once activated, light chain endopeptidase of the toxin is
released from the intracellular vesicle into the cytosol where it
appears to specifically target one of three known core components
of the neurotransmitter release apparatus. These core proteins,
vesicle-associated membrane protein (VAMP)/synaptobrevin,
synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin,
are necessary for synaptic vesicle docking and fusion at the nerve
terminal and constitute members of the soluble
N-ethylmaleimide-sensitive factor-attachment protein-receptor
(SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in the
carboxyl-terminal region, releasing a nine or twenty-six amino acid
segment, respectively, and BoNT/C1 also cleaves SNAP-25 near the
carboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F
and BoNT/G, and tetanus toxin, act on the conserved central portion
of VAMP, and release the amino-terminal portion of VAMP into the
cytosol. BoNT/C1 cleaves syntaxin at a single site near the
cytosolic membrane surface. The selective proteolysis of synaptic
SNAREs accounts for the block of neurotransmitter release caused by
Clostridial toxins in vivo. The SNARE protein targets of
Clostridial toxins are common to exocytosis in a variety of
non-neuronal types; in these cells, as in neurons, light chain
peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et
al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter
Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,
Botulinum and Tetanus Neurotoxins: Structure, Function and
Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002);
Giovanna Lalli et al., The Journey of Tetanus and Botulinum
Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437,
(2003).
[0021] The three-dimensional crystal structures of BoNT/A, BoNT/B
and the H.sub.C domain of TeNT indicate that the three functional
domains of Clostridial neurotoxins are structurally distinct
domains that are shared by all Clostridial toxins. The HEXXH
consensus motif of the light chain forms the tetrahedral zinc
binding pocket of the catalytic site located in a deep cleft on the
protein surface that is accessible by a channel. The structure of
the H.sub.N and H.sub.C domains consists primarily of .beta.-sheet
topologies that are linked by a single .alpha.-helix. The
cylindrical-shaped H.sub.N domain comprises two long amphipathic
.alpha.-helices that resemble the coiled-coil motif found in some
viral proteins. The H.sub.N domain also forms a long unstructured
loop called the `translocation belt,` which wraps around a large
negatively charged cleft of the light chain that blocks access of
the zinc atom to the catalytic-binding pocket of active site. The
H.sub.C domain comprises two distinct structural features of
roughly equal size that indicate function. The first, designated
the H.sub.CN domain, is located in the amino half of the H.sub.C
domain. The H.sub.CN domain forms a .beta.-barrel, jelly-roll fold.
The H.sub.CC domain is the second domain that comprises the H.sub.C
domain. This carboxyl-terminal domain comprises a modified
.beta.-trefoil domain which forms three distinct carbohydrate
binding regions that resembles the carbohydrate binding moiety
found in many sugar-binding proteins, such as, e.g., serum amyloid
P, sialidase, cryia, insecticidal .differential.-endotoxin and
lectins. Biochemical studies indicate that the .beta.-trefoil
domain structure of the H.sub.CC domain appears to mediate the
binding to specific carbohydrate containing components of the
Clostridial toxin receptor on the cell surface, see, e.g.,
Krzysztof Ginalski et al., Structure-based Sequence Alignment for
the Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family
Provides Residue Level Information About the Putative Ganglioside
Binding Site, 482(1-2) FEBS Lett. 119-124 (2000). The H.sub.C
domain tilts away from the H.sub.N domain exposing the surface
loops and making them accessible for binding. No contacts occur
between the light chain and the H.sub.C domain.
[0022] Aspects of the present specification provide, in part, a
Clostridial toxin. As used herein, the term "Clostridial toxin"
refers to any neurotoxin produced by a Clostridial toxin strain
that can execute the overall cellular mechanism whereby a
Clostridial toxin intoxicates a cell and encompasses the binding of
a Clostridial toxin to a low or high affinity receptor complex, the
internalization of the toxin/receptor complex, the translocation of
the Clostridial toxin light chain into the cytoplasm and the
enzymatic modification of a Clostridial toxin substrate. A
Clostridial toxin comprises a Clostridial toxin enzymatic domain, a
Clostridial toxin translocation domain, and a Clostridial toxin
binding domain. Exemplary Clostridial toxins include those produced
by a Clostridium botulinum, a Clostridium tetani, a Clostridium
baratii and a Clostridium butyricum.
[0023] A Clostridial toxin includes, without limitation, naturally
occurring Clostridial toxin variants, such as, e.g., Clostridial
toxin isoforms and Clostridial toxin subtypes; non-naturally
occurring Clostridial toxin variants, such as, e.g., conservative
Clostridial toxin variants, non-conservative Clostridial toxin
variants, and active Clostridial toxin fragments thereof, or any
combination thereof. As used herein, the term "Clostridial toxin
variant," whether naturally-occurring or non-naturally-occurring,
refers to a Clostridial toxin that has at least one amino acid
change from the corresponding region of the disclosed reference
sequences (Table 1) and can be described in percent identity to the
corresponding region of that reference sequence. As non-limiting
examples, a BoNT/A variant of SEQ ID NO: 1 will have at least one
amino acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as compared to the corresponding position(s)
of SEQ ID NO: 1; a BoNT/B variant of SEQ ID NO: 6 will have at
least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to the
corresponding position(s) of SEQ ID NO: 6; a BoNT/C1 variant of SEQ
ID NO: 11 will have at least one amino acid difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared
to the corresponding position(s) of SEQ ID NO: 11; a BoNT/D variant
of SEQ ID NO: 13 will have at least one amino acid difference, such
as, e.g., an amino acid substitution, deletion or addition, as
compared to the corresponding position(s) of SEQ ID NO: 13; a
BoNT/E variant of SEQ ID NO: 15 will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the corresponding position(s) of SEQ ID
NO: 15; a BoNT/F variant of SEQ ID NO: 18 will have at least one
amino acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as compared to the corresponding position(s)
of SEQ ID NO: 18; a BoNT/G variant of SEQ ID NO: 21 will have at
least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to the
corresponding position(s) of SEQ ID NO: 21; a TeNT variant c of SEQ
ID NO: 22 will have at least one amino acid difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared
to the corresponding position(s) of SEQ ID NO: 22; a BaNT variant
of SEQ ID NO: 23 will have at least one amino acid difference, such
as, e.g., an amino acid substitution, deletion or addition, as
compared to the corresponding position(s) of SEQ ID NO: 23; and a
BuNT variant of SEQ ID NO: 24 will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the corresponding position(s) of SEQ ID
NO: 24.
[0024] As used herein, the term "naturally occurring Clostridial
toxin variant" refers to any Clostridial toxin produced without the
aid of any human manipulation, including, without limitation,
Clostridial toxin isoforms produced from alternatively-spliced
transcripts, Clostridial toxin isoforms produced by spontaneous
mutation and Clostridial toxin subtypes. Non-limiting examples of a
Clostridial toxin isoform include, e.g., BoNT/A isoforms, BoNT/B
isoforms, BoNT/C1 isoforms, BoNT/D isoforms, BoNT/E isoforms,
BoNT/F isoforms, BoNT/G isoforms, TeNT isoforms, BaNT isoforms and
BuNT isoforms. Non-limiting examples of a Clostridial toxin subtype
include, e.g., BoNT/A subtypes BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4,
and BoNT/A5; BoNT/B subtypes BoNT/B1, BoNT/B2, BoNT/B3, BoNT/B
bivalent and BoNT/B nonproteolytic; BoNT/C1 subtypes BoNT/C1-1 and
BoNT/C1-2; BoNT/E subtypes BoNT/E1, BoNT/E2, and BoNT/E3; BoNT/F
subtypes BoNT/F1, BoNT/F2, and BoNT/F3; and BuNT subtypes BuNT-1,
and BuNT-2. Other non-limiting examples of a Clostridial toxin
subtype include, e.g., BoNT/A subtypes SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; BoNT/B subtypes SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:
10; BoNT/C1 subtypes SEQ ID NO: 11 and SEQ ID NO: 12; BoNT/E
subtypes SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; BoNT/F
subtypes SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; and BuNT
subtypes SEQ ID NO: 24 and SEQ ID NO: 25.
[0025] As used herein, the term "non-naturally occurring
Clostridial toxin variant" refers to any Clostridial toxin produced
with the aid of human manipulation, including, without limitation,
Clostridial toxins produced by genetic engineering using random
mutagenesis or rational design and Clostridial toxins produced by
chemical synthesis. Non-limiting examples of non-naturally
occurring Clostridial toxin variants include, e.g., conservative
Clostridial toxin variants, non-conservative Clostridial toxin
variants, and active Clostridial toxin fragments.
[0026] As used herein, the term "conservative Clostridial toxin
variant" refers to a Clostridial toxin that has at least one amino
acid substituted by another amino acid or an amino acid analog that
has at least one property similar to that of the original amino
acid from the reference Clostridial toxin sequence (Table 1).
Examples of properties include, without limitation, similar size,
topography, charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity, hydrogen-bonding capacity, a
physicochemical property, of the like, or any combination thereof.
A conservative Clostridial toxin variant can function in
substantially the same manner as the reference Clostridial toxin on
which the conservative Clostridial toxin variant is based, and can
be substituted for the reference Clostridial toxin in any aspect of
the present specification. A conservative Clostridial toxin variant
may substitute 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 200,
300, 400, or 500 or more amino acids from the reference Clostridial
toxin on which the conservative Clostridial toxin variant is based.
A conservative Clostridial toxin variant can also substitute at
least 5, 10, 15, 20, or 25 contiguous amino acids from the
reference Clostridial toxin on which the conservative Clostridial
toxin variant is based. Non-limiting examples of a conservative
Clostridial toxin variant include, e.g., conservative BoNT/A
variants, conservative BoNT/B variants, conservative BoNT/C1
variants, conservative BoNT/D variants, conservative BoNT/E
variants, conservative BoNT/F variants, conservative BoNT/G
variants, conservative TeNT variants, conservative BaNT variants
and conservative BuNT variants.
[0027] As used herein, the term "non-conservative Clostridial toxin
variant" refers to a Clostridial toxin in which 1) at least one
amino acid is deleted from the reference Clostridial toxin on which
the non-conservative Clostridial toxin variant is based; 2) at
least one amino acid added to the reference Clostridial toxin on
which the non-conservative Clostridial toxin is based; or 3) at
least one amino acid is substituted by another amino acid or an
amino acid analog that does not share any property similar to that
of the original amino acid from the reference Clostridial toxin
sequence (Table 1). A non-conservative Clostridial toxin variant
can function in substantially the same manner as the reference
Clostridial toxin on which the non-conservative Clostridial toxin
variant is based, and can be substituted for the reference
Clostridial toxin in any aspect of the present specification. A
non-conservative Clostridial toxin variant can delete one or more
amino acids, two or more amino acids, three or more amino acids,
four or more amino acids, five or more amino acids, and ten or more
amino acids from the reference Clostridial toxin on which the
non-conservative Clostridial toxin variant is based. A
non-conservative Clostridial toxin variant can add one or more
amino acids, two or more amino acids, three or more amino acids,
four or more amino acids, five or more amino acids, and ten or more
amino acids to the reference Clostridial toxin on which the
non-conservative Clostridial toxin variant is based. A
non-conservative Clostridial toxin variant may substitute 1, 2, 3,
4, 5, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, or 500 or more
amino acids from the reference Clostridial toxin on which the
non-conservative Clostridial toxin variant is based. A
non-conservative Clostridial toxin variant can also substitute at
least 5, 10, 15, 20, or 25 contiguous amino acids from the
reference Clostridial toxin on which the non-conservative
Clostridial toxin variant is based. Non-limiting examples of a
non-conservative Clostridial toxin variant include, e.g.,
non-conservative BoNT/A variants, non-conservative BoNT/B variants,
non-conservative BoNT/C1 variants, non-conservative BoNT/D
variants, non-conservative BoNT/E variants, non-conservative BoNT/F
variants, non-conservative BoNT/G variants, non-conservative TeNT
variants, non-conservative BaNT variants and non-conservative BuNT
variants.
[0028] It is also envisioned that any of a variety of Clostridial
toxin fragments can be useful in aspects of the present
specification with the proviso that these active fragments can
execute the overall cellular mechanism whereby a Clostridial toxin
proteolytically cleaves a substrate. Thus, aspects of this
embodiment can include Clostridial toxin fragments having a length
of, e.g., at least 600, 700, 800, 900, 1000, 1100, or at least 1200
amino acids. Other aspects of this embodiment, can include
Clostridial toxin fragments having a length of, e.g., at most 600,
700, 800, 900, 1000, 1100, or at most 1200 amino acids.
[0029] It is also envisioned that any of a variety of Clostridial
toxin fragments comprising the light chain can be useful in aspects
of the present specification with the proviso that these light
chain fragments can specifically target the core components of the
neurotransmitter release apparatus and thus participate in
executing the overall cellular mechanism whereby a Clostridial
toxin proteolytically cleaves a substrate. The light chains of
Clostridial toxins are approximately 420-460 amino acids in length
and comprise a Clostridial toxin enzymatic domain (Table 1).
Research has shown that the entire length of a Clostridial toxin
light chain is not necessary for the enzymatic activity of the
Clostridial toxin enzymatic domain. As a non-limiting example, the
first eight amino acids of a BoNT/A light chain are not required
for enzymatic activity. As another non-limiting example, the first
eight amino acids of the TeNT light chain are not required for
enzymatic activity. Likewise, the carboxyl-terminus of the light
chain is not necessary for activity. As a non-limiting example, the
last 32 amino acids of the BoNT/A light chain are not required for
enzymatic activity. As another non-limiting example, the last 31
amino acids of the TeNT light chain are not required for enzymatic
activity. Thus, aspects of this embodiment include a Clostridial
toxin light chain comprising a Clostridial toxin enzymatic domain
having a length of, e.g., at least 350, 375, 400, 425, or 450 amino
acids. Other aspects of this embodiment include a Clostridial toxin
light chain comprising a Clostridial toxin enzymatic domain having
a length of, e.g., at most 350, 375, 400, 425, or 450 amino
acids.
[0030] It is also envisioned that any of a variety of Clostridial
toxin H.sub.N regions comprising a Clostridial toxin translocation
domain can be useful in aspects of the present specification with
the proviso that these active fragments can facilitate the release
of the LC from intracellular vesicles into the cytoplasm of the
target cell and thus participate in executing the overall cellular
mechanism whereby a Clostridial toxin proteolytically cleaves a
substrate. The H.sub.N regions from the heavy chains of Clostridial
toxins are approximately 410-430 amino acids in length and comprise
a Clostridial toxin translocation domain (Table 1). Research has
shown that the entire length of a H.sub.N region from a Clostridial
toxin heavy chain is not necessary for the translocating activity
of the Clostridial toxin translocation domain. Thus, aspects of
this embodiment can include Clostridial toxin H.sub.N regions
comprising a Clostridial toxin translocation domain having a length
of, e.g., at least 350, 375, 400, or 425 amino acids. Other aspects
of this embodiment can include Clostridial toxin H.sub.N regions
comprising Clostridial toxin translocation domain having a length
of, e.g., at most 350, 375, 400, or 425 amino acids.
[0031] It is also envisioned that any of a variety of Clostridial
toxin H.sub.C regions comprising a Clostridial toxin binding domain
can be useful in aspects of the present specification with the
proviso that these active fragments can determine the binding
activity and binding specificity of the toxin to the receptor
complex located at the surface of the target cell and facilitate
the overall cellular mechanism whereby a Clostridial toxin
proteolytically cleaves a substrate. The H.sub.C regions from the
heavy chains of Clostridial toxins are approximately 400-440 amino
acids in length and comprise a binding domain (Table 1). Research
has shown that the entire length of a H.sub.C region from a
Clostridial toxin heavy chain is not necessary for the binding
activity of the Clostridial toxin binding domain. Thus, aspects of
this embodiment can include Clostridial toxin H.sub.C regions
comprising a binding domain having a length of, e.g., at least 350,
375, 400, or 425 amino acids. Other aspects of this embodiment can
include Clostridial toxin H.sub.C regions comprising a binding
domain having a length of, e.g., at most 350, 375, 400, or 425
amino acids.
[0032] Any of a variety of sequence alignment methods can be used
to determine percent identity, including, without limitation,
global methods, local methods and hybrid methods, such as, e.g.,
segment approach methods. Protocols to determine percent identity
are routine procedures within the scope of one skilled in the art
and from the teaching herein.
[0033] Global methods align sequences from the beginning to the end
of the molecule and determine the best alignment by adding up
scores of individual residue pairs and by imposing gap penalties.
Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D.
Thompson et al., CLUSTAL W: Improving the Sensitivity of
Progressive Multiple Sequence Alignment Through Sequence Weighting,
Position-Specific Gap Penalties and Weight Matrix Choice, 22(22)
Nucleic Acids Research 4673-4680 (1994); and iterative refinement,
see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of
Multiple Protein Sequence Alignments by Iterative Refinement as
Assessed by Reference to Structural Alignments, 264(4) J. Mol.
Biol. 823-838 (1996).
[0034] Local methods align sequences by identifying one or more
conserved motifs shared by all of the input sequences. Non-limiting
methods include, e.g., Match-box, see, e.g., Eric Depiereux and
Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the
Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS
501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al.,
Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for
Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see,
e.g., Ivo Van Walle et al., Align-M--A New Algorithm for Multiple
Alignment of Highly Divergent Sequences, 20(9) Bioinformatics,
1428-1435 (2004).
[0035] Hybrid methods combine functional aspects of both global and
local alignment methods. Non-limiting methods include, e.g.,
segment-to-segment comparison, see, e.g., Burkhard Morgenstern et
al., Multiple DNA and Protein Sequence Alignment Based On
Segment-To-Segment Comparison, 93(22) Proc. Natl. Acad. Sci. U.S.A.
12098-12103 (1996); T-Coffee, see, e.g., Cedric Notredame et al.,
T-Coffee: A Novel Algorithm for Multiple Sequence Alignment, 302(1)
J. Mol. Biol. 205-217 (2000); MUSCLE, see, e.g., Robert C. Edgar,
MUSCLE: Multiple Sequence Alignment With High Score Accuracy and
High Throughput, 32(5) Nucleic Acids Res. 1792-1797 (2004); and
DIALIGN-T, see, e.g., Amarendran R Subramanian et al., DIALIGN-T:
An Improved Algorithm for Segment-Based Multiple Sequence
Alignment, 6(1) BMC Bioinformatics 66 (2005).
[0036] The present specification describes various polypeptide
variants where one amino acid is substituted for another, such as,
e.g., Clostridial toxin variants, Clostridial toxin enzymatic
domain variants, Clostridial toxin translocation domain variants,
Clostridial toxin binding domain variants, non-Clostridial toxin
binding domain variants, and protease cleavage site variants. A
substitution can be assessed by a variety of factors, such as,
e.g., the physical properties of the amino acid being substituted
(Table 2) or how the original amino acid would tolerate a
substitution (Table 3). The selections of which amino acid can be
substituted for another amino acid in a polypeptide are known to a
person of ordinary skill in the art.
TABLE-US-00002 TABLE 2 Amino Acid Properties Property Amino Acids
Aliphatic G, A, I, L, M, P, V Aromatic F, H, W, Y C-beta branched
I, V, T Hydrophobic C, F, I, L, M, V, W Small polar D, N, P Small
non-polar A, C, G, S, T Large polar E, H, K, Q, R, W, Y Large
non-polar F, I, L, M, V Charged D, E, H, K, R Uncharged C, S, T
Negative D, E Positive H, K, R Acidic D, E Basic K, R Amide N,
Q
TABLE-US-00003 TABLE 3 Amino Acid Substitutions Amino Acid Favored
Substitution Neutral Substitutions Disfavored substitution A G, S,
T C, E, I, K, M, L, P, Q, R, V D, F, H, N, Y, W C F, S, Y, W A, H,
I, M, L, T, V D, E, G, K, N, P, Q, R D E, N G, H, K, P, Q, R, S, T
A, C, I, L, E D, K, Q A, H, N, P, R, S, T C, F, G, I, L, M, V, W, Y
F M, L, W, Y C, I, V A, D, E, G, H, K, N, P, Q, R, S, T G A, S D,
K, N, P, Q, R C, E, F, H, I, L, M, T, V, W, Y H N, Y C, D, E, K, Q,
R, S, T, W A, F, G, I, L, M, P, V I V, L, M A, C, T, F, Y D, E, G,
H, K, N, P, Q, R, S, W K Q, E, R A, D, G, H, M, N, P, S, T C, F, I,
L, V, W, Y L F, I, M, V A, C, W, Y D, E, G, H, K, N, P, Q, R, S, T
M F, I, L, V A, C, R, Q, K, T, W, Y D, E, G, H, N, P, S N D, H, S
E, G, K, Q, R, T A, C, F, I, L, M, P, V, W, Y P -- A, D, E, G, K,
Q, R, S, T C, F, H, I, L, M, N, V, W, Y Q E, K, R A, D, G, H, M, N,
P, S, T C, F, I, L, V, W, Y R K, Q A, D, E, G, H, M, N, P, S, T C,
F, I, L, V, W, Y S A, N, T C, D, E, G, H, K, P, Q, R, T F, I, L, M,
V, W, Y T S A, C, D, E, H, I, K, M, N, P, Q, R, V F, G, L, W, Y V
I, L, M A, C, F, T, Y D, E, G, H, K, N, P, Q, R, S, W W F, Y H, L,
M A, C, D, E, G, I, K, N, P, Q, R, S, T, V Y F, H, W C, I, L, M, V
A, D, E, G, K, N, P, Q, R, S, T Matthew J. Betts and Robert, B.
Russell, Amino Acid Properties and Consequences of Substitutions,
pp. 289-316, In Bioinformatics for Geneticists, (eds Michael R.
Barnes, Ian C. Gray, Wiley, 2003).
[0037] Thus, in an embodiment, a Clostridial toxin comprises a
Clostridial toxin enzymatic domain, a Clostridial toxin
translocation domain, and a Clostridial toxin binding domain. In an
aspect of this embodiment, a Clostridial toxin comprises a
naturally occurring Clostridial toxin variant, such as, e.g., a
Clostridial toxin isoform or a Clostridial toxin subtype. In
another aspect of this embodiment, a Clostridial toxin comprises a
non-naturally occurring Clostridial toxin variant, such as, e.g., a
conservative Clostridial toxin variant, a non-conservative
Clostridial toxin variant or an active Clostridial toxin fragment,
or any combination thereof. In another aspect of this embodiment, a
Clostridial toxin comprises a Clostridial toxin enzymatic domain or
an active fragment thereof, a Clostridial toxin translocation
domain or an active fragment thereof, a Clostridial toxin binding
domain or an active fragment thereof, or any combination thereof.
In other aspects of this embodiment, a Clostridial toxin can
comprise a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a
BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT.
[0038] In another embodiment, a hydrophobic amino acid at one
particular position in the polypeptide chain of the Clostridial
toxin can be substituted with another hydrophobic amino acid.
Examples of hydrophobic amino acids include, e.g., C, F, I, L, M, V
and W. In another aspect of this embodiment, an aliphatic amino
acid at one particular position in the polypeptide chain of the
Clostridial toxin can be substituted with another aliphatic amino
acid. Examples of aliphatic amino acids include, e.g., A, I, L, P,
and V. In yet another aspect of this embodiment, an aromatic amino
acid at one particular position in the polypeptide chain of the
Clostridial toxin can be substituted with another aromatic amino
acid. Examples of aromatic amino acids include, e.g., F, H, W and
Y. In still another aspect of this embodiment, a stacking amino
acid at one particular position in the polypeptide chain of the
Clostridial toxin can be substituted with another stacking amino
acid. Examples of stacking amino acids include, e.g., F, H, W and
Y. In a further aspect of this embodiment, a polar amino acid at
one particular position in the polypeptide chain of the Clostridial
toxin can be substituted with another polar amino acid. Examples of
polar amino acids include, e.g., D, E, K, N, Q, and R. In a further
aspect of this embodiment, a less polar or indifferent amino acid
at one particular position in the polypeptide chain of the
Clostridial toxin can be substituted with another less polar or
indifferent amino acid. Examples of less polar or indifferent amino
acids include, e.g., A, H, G, P, S, T, and Y. In a yet further
aspect of this embodiment, a positive charged amino acid at one
particular position in the polypeptide chain of the Clostridial
toxin can be substituted with another positive charged amino acid.
Examples of positive charged amino acids include, e.g., K, R, and
H. In a still further aspect of this embodiment, a negative charged
amino acid at one particular position in the polypeptide chain of
the Clostridial toxin can be substituted with another negative
charged amino acid. Examples of negative charged amino acids
include, e.g., D and E. In another aspect of this embodiment, a
small amino acid at one particular position in the polypeptide
chain of the Clostridial toxin can be substituted with another
small amino acid. Examples of small amino acids include, e.g., A,
D, G, N, P, S, and T. In yet another aspect of this embodiment, a
C-beta branched amino acid at one particular position in the
polypeptide chain of the Clostridial toxin can be substituted with
another C-beta branched amino acid. Examples of C-beta branched
amino acids include, e.g., I, T and V.
[0039] In another embodiment, a Clostridial toxin comprises a
BoNT/A. In an aspect of this embodiment, a BoNT/A comprises a
BoNT/A enzymatic domain, a BoNT/A translocation domain, and a
BoNT/A binding domain. In another aspect of this embodiment, a
BoNT/A comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, or SEQ ID NO: 5. In another aspect of this embodiment, a
BoNT/A comprises a naturally occurring BoNT/A variant, such as,
e.g., a BoNT/A isoform or a BoNT/A subtype. In another aspect of
this embodiment, a BoNT/A comprises a naturally occurring BoNT/A
variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
or SEQ ID NO: 5, such as, e.g., a BoNT/A isoform or a BoNT/A
subtype. In still another aspect of this embodiment, a BoNT/A
comprises a non-naturally occurring BoNT/A variant, such as, e.g.,
a conservative BoNT/A variant, a non-conservative BoNT/A variant or
an active BoNT/A fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/A comprises a
non-naturally occurring BoNT/A variant of SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., a
conservative BoNT/A variant, a non-conservative BoNT/A variant, an
active BoNT/A fragment, or any combination thereof. In yet another
aspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic
domain or an active fragment thereof, a BoNT/A translocation domain
or an active fragment thereof, a BoNT/A binding domain or an active
fragment thereof, or any combination thereof. In yet another aspect
of this embodiment, a BoNT/A comprising a BoNT/A enzymatic domain
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID
NO: 5, or an active fragment thereof, a BoNT/A translocation domain
of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ
ID NO: 5, or an active fragment thereof, a BoNT/A binding domain of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID
NO: 5, or an active fragment thereof, or any combination
thereof.
[0040] In other aspects of this embodiment, a BoNT/A comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or
SEQ ID NO: 5; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to SEQ ID NO: 1, SEQ ID NO: 2, SEQ
ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In yet other aspects of
this embodiment, a BoNT/A comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, or SEQ ID NO: 5; at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
In still other aspects of this embodiment, a BoNT/A comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, or SEQ ID NO: 5.
[0041] In another embodiment, a Clostridial toxin comprises a
BoNT/B. In an aspect of this embodiment, a BoNT/B comprises a
BoNT/B enzymatic domain, a BoNT/B translocation domain, and a
BoNT/B binding domain. In another aspect of this embodiment, a
BoNT/B comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 9, or SEQ ID NO: 10. In another aspect of this embodiment, a
BoNT/B comprises a naturally occurring BoNT/B variant, such as,
e.g., a BoNT/B isoform or a BoNT/B subtype. In another aspect of
this embodiment, a BoNT/B comprises a naturally occurring BoNT/B
variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
or SEQ ID NO: 10, such as, e.g., a BoNT/B isoform or a BoNT/B
subtype. In still another aspect of this embodiment, a BoNT/B
comprises a non-naturally occurring BoNT/B variant, such as, e.g.,
a conservative BoNT/B variant, a non-conservative BoNT/B variant,
an active BoNT/B fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/B comprises a
non-naturally occurring BoNT/B variant of SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., a
conservative BoNT/B variant, a non-conservative BoNT/B variant, an
active BoNT/B fragment, or any combination thereof. In yet another
aspect of this embodiment, a BoNT/B comprises a BoNT/B enzymatic
domain or an active fragment thereof, a BoNT/B translocation domain
or active fragment thereof, a BoNT/B binding domain or active
fragment thereof, or any combination thereof. In yet another aspect
of this embodiment, a BoNT/B comprises a BoNT/B enzymatic domain of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID
NO: 10, or active fragment thereof, a BoNT/B translocation domain
of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ
ID NO: 10, or active fragment thereof, a BoNT/B binding domain of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID
NO: 10, or active fragment thereof, or any combination thereof.
[0042] In other aspects of this embodiment, a BoNT/B comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or
SEQ ID NO: 102; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In yet other aspects of
this embodiment, a BoNT/B comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, or SEQ ID NO: 10; at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
In still other aspects of this embodiment, a BoNT/B comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, or SEQ ID NO: 10.
[0043] In another embodiment, a Clostridial toxin comprises a
BoNT/C1. In an aspect of this embodiment, a BoNT/C1 comprises a
BoNT/C1 enzymatic domain, a BoNT/C1 translocation domain, and a
BoNT/C1 binding domain. In another aspect of this embodiment, a
BoNT/C1 comprises SEQ ID NO: 11 or SEQ ID NO: 12. In another aspect
of this embodiment, a BoNT/C1 comprises a naturally occurring
BoNT/C1 variant, such as, e.g., a BoNT/C1 isoform or a BoNT/C1
subtype. In another aspect of this embodiment, a BoNT/C1 comprises
a naturally occurring BoNT/C1 variant of SEQ ID NO: 11 or SEQ ID
NO: 12, such as, e.g., a BoNT/C1 isoform or a BoNT/C1 subtype. In
still another aspect of this embodiment, a BoNT/C1 comprises a
non-naturally occurring BoNT/C1 variant, such as, e.g., a
conservative BoNT/C1 variant, a non-conservative BoNT/C1 variant,
an active BoNT/C1 fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/C1 comprises a
non-naturally occurring BoNT/C1 variant of SEQ ID NO: 11 or SEQ ID
NO: 12, such as, e.g., a conservative BoNT/C1 variant, a
non-conservative BoNT/C1 variant, an active BoNT/C1 fragment, or
any combination thereof. In yet another aspect of this embodiment,
a BoNT/C1 comprises a BoNT/C1 enzymatic domain or active fragment
thereof, a BoNT/C1 translocation domain or active fragment thereof,
a BoNT/C1 binding domain, active fragment thereof, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/C1 comprises a BoNT/C1 enzymatic domain of SEQ ID NO: 11 or
SEQ ID NO: 12, or active fragment thereof, a BoNT/C1 translocation
domain of SEQ ID NO: 11 or SEQ ID NO: 12, or active fragment
thereof, a BoNT/C1 binding domain of SEQ ID NO: 11 or SEQ ID NO:
12, or active fragment thereof, or any combination thereof.
[0044] In other aspects of this embodiment, a BoNT/C1 comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 11 or SEQ ID NO: 12; or at most 70%, at most 75%,
at most 80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO:
11 or SEQ ID NO: 12. In yet other aspects of this embodiment, a
BoNT/C1 comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500
non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 11 or SEQ ID NO: 12; at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 11 or SEQ ID NO: 12. In still
other aspects of this embodiment, a BoNT/C1 comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 3; at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 11 or SEQ ID NO: 12.
[0045] In another embodiment, a Clostridial toxin comprises a
BoNT/D. In an aspect of this embodiment, a BoNT/D comprises a
BoNT/D enzymatic domain, a BoNT/D translocation domain, and a
BoNT/D binding domain. In another aspect of this embodiment, a
BoNT/D comprises SEQ ID NO: 13 or SEQ ID NO: 14. In another aspect
of this embodiment, a BoNT/D comprises a naturally occurring BoNT/D
variant, such as, e.g., a BoNT/D isoform or a BoNT/D subtype. In
another aspect of this embodiment, a BoNT/D comprises a naturally
occurring BoNT/D variant of SEQ ID NO: 13 or SEQ ID NO: 14, such
as, e.g., a BoNT/D isoform or a BoNT/D subtype. In still another
aspect of this embodiment, a BoNT/D comprises a non-naturally
occurring BoNT/D variant, such as, e.g., a conservative BoNT/D
variant, a non-conservative BoNT/D variant, an active BoNT/D
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/D comprises a non-naturally occurring
BoNT/D variant of SEQ ID NO: 13 or SEQ ID NO: 14, such as, e.g., a
conservative BoNT/D variant, a non-conservative BoNT/D variant, an
active BoNT/D fragment, or any combination thereof. In yet another
aspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic
domain or an active fragment thereof, a BoNT/D translocation domain
or an active fragment thereof, a BoNT/D binding domain or an active
fragment thereof, or any combination thereof. In yet another aspect
of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domain of
SEQ ID NO: 13 or SEQ ID NO: 14, or an active fragment thereof, a
BoNT/D translocation domain of SEQ ID NO: 13 or SEQ ID NO: 14, or
an active fragment thereof, a BoNT/D binding domain of SEQ ID NO:
13 or SEQ ID NO: 14, or an active fragment thereof, or any
combination thereof.
[0046] In other aspects of this embodiment, a BoNT/D comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 13 or SEQ ID NO: 14; or at most 70%, at most 75%,
at most 80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO:
13 or SEQ ID NO: 14. In yet other aspects of this embodiment, a
BoNT/D comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500
non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 13 or SEQ ID NO: 14; at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 13 or SEQ ID NO: 14. In still
other aspects of this embodiment, a BoNT/D comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 13 or SEQ ID
NO: 14; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,
200, 300, 400, or 500 contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 13 or SEQ ID NO:
14.
[0047] In another embodiment, a Clostridial toxin comprises a
BoNT/E. In an aspect of this embodiment, a BoNT/E comprises a
BoNT/E enzymatic domain, a BoNT/E translocation domain, and a
BoNT/E binding domain. In another aspect of this embodiment, a
BoNT/E comprises SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In
another aspect of this embodiment, a BoNT/E comprises a naturally
occurring BoNT/E variant, such as, e.g., a BoNT/E isoform or a
BoNT/E subtype. In another aspect of this embodiment, a BoNT/E
comprises a naturally occurring BoNT/E variant of SEQ ID NO: 15,
SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a BoNT/E isoform or
a BoNT/E subtype. In still another aspect of this embodiment, a
BoNT/E comprises a non-naturally occurring BoNT/E variant, such as,
e.g., a conservative BoNT/E variant, a non-conservative BoNT/E
variant, an active BoNT/E fragment, or any combination thereof. In
still another aspect of this embodiment, a BoNT/E comprises a
non-naturally occurring BoNT/E variant of SEQ ID NO: 15, SEQ ID NO:
16, or SEQ ID NO: 17, such as, e.g., a conservative BoNT/E variant,
a non-conservative BoNT/E variant, an active BoNT/E fragment, or
any combination thereof. In yet another aspect of this embodiment,
a BoNT/E comprises a BoNT/E enzymatic domain or an active fragment
thereof, a BoNT/E translocation domain or active fragment thereof,
a BoNT/E binding domain or active fragment thereof, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/E comprises a BoNT/E enzymatic domain of SEQ ID NO: 15, SEQ ID
NO: 16, or SEQ ID NO: 17, or active fragment thereof, a BoNT/E
translocation domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:
17, or active fragment thereof, a BoNT/E binding domain of SEQ ID
NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, or active fragment
thereof, or any combination thereof.
[0048] In other aspects of this embodiment, a BoNT/E comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or at most
70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most
95% to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In yet other
aspects of this embodiment, a BoNT/E comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 15, SEQ ID
NO: 16, or SEQ ID NO: 17; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In still other aspects of
this embodiment, a BoNT/E comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID
NO: 17; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,
200, 300, 400, or 500 contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 15, SEQ ID NO: 16, or
SEQ ID NO: 17.
[0049] In another embodiment, a Clostridial toxin comprises a
BoNT/F. In an aspect of this embodiment, a BoNT/F comprises a
BoNT/F enzymatic domain, a BoNT/F translocation domain, and a
BoNT/F binding domain. In another aspect of this embodiment, a
BoNT/F comprises SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In
another aspect of this embodiment, a BoNT/F comprises a naturally
occurring BoNT/F variant, such as, e.g., a BoNT/F isoform or a
BoNT/F subtype. In another aspect of this embodiment, a BoNT/F
comprises a naturally occurring BoNT/F variant of SEQ ID NO: 18,
SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a BoNT/F isoform or
a BoNT/F subtype. In still another aspect of this embodiment, a
BoNT/F comprises a non-naturally occurring BoNT/F variant, such as,
e.g., a conservative BoNT/F variant, a non-conservative BoNT/F
variant, an active BoNT/F fragment, or any combination thereof. In
still another aspect of this embodiment, a BoNT/F comprises a
non-naturally occurring BoNT/F variant of SEQ ID NO: 18, SEQ ID NO:
19, or SEQ ID NO: 20, such as, e.g., a conservative BoNT/F variant,
a non-conservative BoNT/F variant, an active BoNT/F fragment, or
any combination thereof. In yet another aspect of this embodiment,
a BoNT/F comprises a BoNT/F enzymatic domain or active fragment
thereof, a BoNT/F translocation domain or active fragment thereof,
a BoNT/F binding domain or active fragment thereof, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/F comprises a BoNT/F enzymatic domain of SEQ ID NO: 18, SEQ ID
NO: 19, or SEQ ID NO: 20, or active fragment thereof, a BoNT/F
translocation domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:
20, or active fragment thereof, a BoNT/F binding domain of SEQ ID
NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, or active fragment
thereof, or any combination thereof.
[0050] In other aspects of this embodiment, a BoNT/F comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; or at most
70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most
95% to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In yet other
aspects of this embodiment, a BoNT/F comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 18, SEQ ID
NO: 19, or SEQ ID NO: 20; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In still other aspects of
this embodiment, a BoNT/F comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID
NO: 20; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,
200, 300, 400, or 500 contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 18, SEQ ID NO: 19, or
SEQ ID NO: 20.
[0051] In another embodiment, a Clostridial toxin comprises a
BoNT/G. In an aspect of this embodiment, a BoNT/G comprises a
BoNT/G enzymatic domain, a BoNT/G translocation domain, and a
BoNT/G binding domain. In another aspect of this embodiment, a
BoNT/G comprises SEQ ID NO: 21. In another aspect of this
embodiment, a BoNT/G comprises a naturally occurring BoNT/G
variant, such as, e.g., a BoNT/G isoform or a BoNT/G subtype. In
another aspect of this embodiment, a BoNT/G comprises a naturally
occurring BoNT/G variant of SEQ ID NO: 21, such as, e.g., a BoNT/G
isoform or a BoNT/G subtype of SEQ ID NO: 21. In still another
aspect of this embodiment, a BoNT/G comprises a non-naturally
occurring BoNT/G variant, such as, e.g., a conservative BoNT/G
variant, a non-conservative BoNT/G variant or an active BoNT/G
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/D comprises a non-naturally occurring
BoNT/G variant of SEQ ID NO: 21, such as, e.g., a conservative
BoNT/G variant, a non-conservative BoNT/G variant, an active BoNT/G
fragment, or any combination thereof. In yet another aspect of this
embodiment, a BoNT/G comprises a BoNT/G enzymatic domain or an
active fragment thereof, a BoNT/G translocation domain or an active
fragment thereof, a BoNT/G binding domain or an active fragment
thereof, or any combination thereof. In yet another aspect of this
embodiment, a BoNT/G comprises a BoNT/G enzymatic domain of SEQ ID
NO: 21 or an active fragment thereof, a BoNT/G translocation domain
of SEQ ID NO: 21 or an active fragment thereof, a BoNT/G binding
domain of SEQ ID NO: 21 or an active fragment thereof, or any
combination thereof.
[0052] In other aspects of this embodiment, a BoNT/G comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 21; or at most 70%, at most 75%, at most 80%, at
most 85%, at most 90%, or at most 95% to SEQ ID NO: 21. In yet
other aspects of this embodiment, a BoNT/G comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 21; at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 21. In still other aspects of
this embodiment, a BoNT/G comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 21; at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous
amino acid deletions, additions, and/or substitutions relative to
SEQ ID NO: 21.
[0053] In another embodiment, a Clostridial toxin comprises a TeNT.
In an aspect of this embodiment, a TeNT comprises a TeNT enzymatic
domain, a TeNT translocation domain, and a TeNT binding domain. In
an aspect of this embodiment, a TeNT comprises SEQ ID NO: 22. In
another aspect of this embodiment, a TeNT comprises a naturally
occurring TeNT variant, such as, e.g., a TeNT isoform or a TeNT
subtype. In another aspect of this embodiment, a TeNT comprises a
naturally occurring TeNT variant of SEQ ID NO: 22, such as, e.g., a
TeNT isoform or a TeNT subtype. In still another aspect of this
embodiment, a TeNT comprises a non-naturally occurring TeNT
variant, such as, e.g., a conservative TeNT variant, a
non-conservative TeNT variant, an active TeNT fragment, or any
combination thereof. In still another aspect of this embodiment, a
TeNT comprises a non-naturally occurring TeNT variant of SEQ ID NO:
22, such as, e.g., a conservative TeNT variant, a non-conservative
TeNT variant, an active TeNT fragment, or any combination thereof.
In yet another aspect of this embodiment, a TeNT comprises a TeNT
enzymatic domain or an active fragment thereof, a TeNT
translocation domain or active fragment thereof, a TeNT binding
domain or active fragment thereof, or any combination thereof. In
yet another aspect of this embodiment, a TeNT comprises a TeNT
enzymatic domain of SEQ ID NO: 22 or active fragment thereof, a
TeNT translocation domain of SEQ ID NO: 22 or active fragment
thereof, a TeNT binding domain of SEQ ID NO: 22 or active fragment
thereof, or any combination thereof.
[0054] In other aspects of this embodiment, a TeNT comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 22; or at most 70%, at most 75%, at most 80%, at
most 85%, at most 90%, or at most 95% to SEQ ID NO: 22. In yet
other aspects of this embodiment, a TeNT comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 22; at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 22. In still other aspects of
this embodiment, a TeNT comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 22; at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous
amino acid deletions, additions, and/or substitutions relative to
SEQ ID NO: 22.
[0055] In another embodiment, a Clostridial toxin comprises a BaNT.
In an aspect of this embodiment, a BaNT comprises a BaNT enzymatic
domain, a BaNT translocation domain, and a BaNT binding domain. In
another aspect of this embodiment, a BaNT comprises SEQ ID NO: 23.
In another aspect of this embodiment, a BaNT comprises a naturally
occurring BaNT variant, such as, e.g., a BaNT isoform or a BaNT
subtype. In another aspect of this embodiment, a BaNT comprises a
naturally occurring BaNT variant of SEQ ID NO: 23, such as, e.g., a
BaNT isoform or a BaNT subtype. In still another aspect of this
embodiment, a BaNT comprises a non-naturally occurring BaNT
variant, such as, e.g., a conservative BaNT variant, a
non-conservative BaNT variant or an active BaNT fragment, or any
combination thereof. In still another aspect of this embodiment, a
BaNT comprises a non-naturally occurring BaNT variant of SEQ ID NO:
23, such as, e.g., a conservative BaNT variant, a non-conservative
BaNT variant, an active BaNT fragment, or any combination thereof.
In yet another aspect of this embodiment, a BaNT comprises a BaNT
enzymatic domain or an active fragment thereof, a BaNT
translocation domain or an active fragment thereof, a BaNT binding
domain or an active fragment thereof, or any combination thereof.
In yet another aspect of this embodiment, a BaNT comprises a BaNT
enzymatic domain of SEQ ID NO: 23 or an active fragment thereof, a
BaNT translocation domain of SEQ ID NO: 23 or an active fragment
thereof, a BaNT binding domain of SEQ ID NO: 23 or an active
fragment thereof, or any combination thereof.
[0056] In other aspects of this embodiment, a BaNT comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 23; or at most 70%, at most 75%, at most 80%, at
most 85%, at most 90%, or at most 95% to SEQ ID NO: 23. In yet
other aspects of this embodiment, a BaNT comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 23; at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 23. In still other aspects of
this embodiment, a BaNT comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,
400, or 500 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 23; at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous
amino acid deletions, additions, and/or substitutions relative to
SEQ ID NO: 23.
[0057] In another embodiment, a Clostridial toxin comprises a BuNT.
In an aspect of this embodiment, a BuNT comprises a BuNT enzymatic
domain, a BuNT translocation domain, and a BuNT binding domain. In
another aspect of this embodiment, a BuNT comprises SEQ ID NO: 24
or SEQ ID NO: 25. In another aspect of this embodiment, a BuNT
comprises a naturally occurring BuNT variant, such as, e.g., a BuNT
isoform or a BuNT subtype. In another aspect of this embodiment, a
BuNT comprises a naturally occurring BuNT variant of SEQ ID NO: 24
or SEQ ID NO: 25, such as, e.g., a BuNT isoform or a BuNT subtype.
In still another aspect of this embodiment, a BuNT comprises a
non-naturally occurring BuNT variant, such as, e.g., a conservative
BuNT variant, a non-conservative BuNT variant, an active BuNT
fragment, or any combination thereof. In still another aspect of
this embodiment, a BuNT comprises a non-naturally occurring BuNT
variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g., a
conservative BuNT variant, a non-conservative BuNT variant, an
active BuNT fragment, or any combination thereof. In yet another
aspect of this embodiment, a BuNT comprises a BuNT enzymatic domain
or an active fragment thereof, a BuNT translocation domain or an
active fragment thereof, a BuNT binding domain, an active fragment
thereof, or any combination thereof. In yet another aspect of this
embodiment, a BuNT comprises a BuNT enzymatic domain of SEQ ID NO:
24 or SEQ ID NO: 25, or an active fragment thereof, a BuNT
translocation domain of SEQ ID NO: 24 or SEQ ID NO: 25, or an
active fragment thereof, a BuNT binding domain of SEQ ID NO: 24 or
SEQ ID NO: 25, or an active fragment thereof, or any combination
thereof.
[0058] In other aspects of this embodiment, a BuNT comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 24 or SEQ ID NO: 25; or at most 70%, at most 75%,
at most 80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO:
24 or SEQ ID NO: 25. In yet other aspects of this embodiment, a
BuNT comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500
non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 24 or SEQ ID NO: 25; at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400,
or 500 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 24 or SEQ ID NO: 25. In still
other aspects of this embodiment, a BuNT comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 100, 200, 300, 400, or 500 contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 24 or SEQ ID
NO: 25; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,
200, 300, 400, or 500 contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 24 or SEQ ID NO:
25.
[0059] As used herein, the term "Clostridial toxin chimeric" or
"Clostridial toxin chimeras" refers to a molecule comprising at
least a portion from one Clostridial toxin and a portion from at
least one other protein to form a toxin with at least one property
different from the reference Clostridial toxins of Table 1.
Non-limiting examples of Clostridial toxin chimeras include a
Clostridial toxin comprising a non-Clostridial toxin enzymatic
domain, a Clostridial toxin comprising a non-Clostridial toxin
translocation domain, a Clostridial toxin comprising a
non-Clostridial toxin binding domain, or any combination thereof.
Other non-limiting example of a Clostridial toxin chimeras include
a Clostridial toxin comprising a enzymatic domain from a different
Clostridial toxin, a Clostridial toxin comprising a translocation
domain from a different Clostridial toxin, a Clostridial toxin
comprising a binding domain from a different Clostridial toxin, or
any combination thereof.
[0060] One class of Clostridial toxin chimeric comprises a modified
Clostridial toxin were the enzymatic domain or portion thereof,
translocation domain or portion thereof, and/or binding domain or
portion thereof of a naturally-occurring Clostridial toxin is
either modified or replaced with an enzymatic domain or portion
thereof, translocation domain or portion thereof, and/or binding
domain or portion thereof of a different Clostridial toxin. As
non-limiting example, the binding domain of BoNT/A can be replaced
with the binding domain of BoNT/B producing a Clostridial toxin
chimeric comprising a BoNT/A enzymatic domain, a BoNT/A
translocation domain, and a BoNT/B binding domain. Such Clostridial
toxin chimeras are described in, e.g., J. Oliver Dolly et al.,
Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,132,259, which
is incorporated by reference in its entirety. As another
non-limiting example, the leucine motif from BoNT/A can be inserted
into the light chain of a BoNT/E in order to increase biological
persistence. Such Clostridial toxin chimeras are described in,
e.g., Lance E. Steward et al., Leucine-based Motif and Clostridial
Toxins, U.S. Patent Publication 2003/0027752 (Feb. 6, 2003); Lance
E. Steward et al., Clostridial Neurotoxin Compositions and Modified
Clostridial Neurotoxins, U.S. Patent Publication 2003/0219462 (Nov.
27, 2003); and Lance E. Steward et al., Clostridial Neurotoxin
Compositions and Modified Clostridial Neurotoxins, U.S. Patent
Publication 2004/0220386 (Nov. 4, 2004), each of which is
incorporated by reference in its entirety.
[0061] Another class of Clostridial toxin chimeric comprises a
Clostridial toxin where the binding domain of a naturally-occurring
Clostridial toxin is either modified or replaced with a binding
domain of a non-Clostridial toxin. Such Clostridial toxin chimeras
possesses an altered cell binding activity because the modified
toxin can either, e.g., 1) use the same receptor present on the
surface of a naturally occurring Clostridial toxin target cell as
that used by the naturally-occurring Clostridial toxin, referred to
as an enhanced cell binding activity for a naturally-occurring
Clostridial toxin target cell; 2) use a different receptor present
on the surface of a naturally occurring Clostridial toxin target
cell, referred to as an altered cell binding activity for a
naturally-occurring Clostridial toxin target cell; or 3) use a
different receptor present on the surface of the non-Clostridial
toxin target cell, referred to as an altered cell binding activity
for a non-naturally-occurring Clostridial toxin target cell, a
re-targeted toxin or a TVEMP.
[0062] A Clostridial toxin chimeric can be a Clostridial toxin with
an enhanced cell binding activity capable of intoxicating a
naturally occurring Clostridial toxin target cell, e.g., a motor
neuron. One way this enhanced binding activity is achieved by
modifying the endogenous binding domain of a naturally-occurring
Clostridial toxin in order to enhance a cell binding activity of
the toxin for its naturally-occurring receptor. Such modifications
to a targeting domain result in, e.g., a enhanced cell binding
activity that increases binding affinity for an endogenous
Clostridial toxin receptor present on a naturally-occurring
Clostridial toxin target cell; an enhanced cell binding activity
that increases binding specificity for a subgroup of endogenous
Clostridial toxin receptors present on a naturally-occurring
Clostridial toxin target cell; or an enhanced cell binding activity
that increases both binding affinity and binding specificity.
Non-limiting examples of modified Clostridial toxins an enhanced
cell binding activity for a naturally-occurring Clostridial toxin
receptor are described in, e.g., Lance E. Steward et al., Modified
Clostridial Toxins with Enhanced Targeting Capabilities For
Endogenous Clostridial Toxin Receptor Systems, U.S. Patent
Publication 2008/0096248; Lance E. Steward, Modified Clostridial
Toxins with Enhanced Translocation Capabilities and Enhanced
Targeting Activity for Clostridial Toxin Target Cells,
International Patent Publication 2008/105901; each of which is
hereby incorporated by reference in its entirety.
[0063] A Clostridial toxin chimeric can be a Clostridial toxin with
an altered cell binding activity capable of intoxicating a
naturally occurring Clostridial toxin target cell, e.g., a motor
neuron. One way this altered capability is achieved by replacing
the endogenous binding domain of a naturally-occurring Clostridial
toxin with a binding domain of another molecule that preferentially
binds to a different receptor present on the surface of a
Clostridial toxin target cell. Such a modification to a binding
domain results in a modified toxin that is able to preferentially
bind to a non-Clostridial toxin receptor present on a Clostridial
toxin target cell. This enhanced binding activity for a naturally
occurring Clostridial toxin target cell allows for lower effective
doses of a modified Clostridial toxin to be administered to an
individual because more toxin will be delivered to the target cell.
Thus, modified Clostridial toxins with an enhanced binding activity
will reduce the undesirable dispersal of the toxin to areas not
targeted for treatment, thereby reducing or preventing the
undesirable side-effects associated with diffusion of a Clostridial
toxin to an unwanted location. Non-limiting examples of modified
Clostridial toxins with an altered cell binding capability for a
Clostridial toxin target cell are described in, e.g., Lance E.
Steward et al., Multivalent Clostridial Toxin Derivatives and
Methods of Their Use, U.S. Pat. No. 7,514,088; Lance E. Steward et
al., Modified Clostridial Toxins with Altered Targeting
Capabilities For Clostridial Toxin Target Cells, U.S. Patent
Publication 2008/0161543; Lance E. Steward, Modified Clostridial
Toxins with Enhanced Translocation Capabilities and Altered
Targeting Activity for Clostridial Toxin Target Cells, U.S. Patent
Publication 2008/0241881; Lance E. Steward et al., Multivalent
Clostridial Toxin Derivatives and Methods of Their Use, U.S. Patent
Publication 2009/0048431; Lance E. Steward et al., Modified
Clostridial Toxins with Altered Targeting Capabilities For
Clostridial Toxin Target Cells, International Patent Publication WO
2007/106115; each of which is hereby incorporated by reference in
its entirety.
[0064] A Clostridial toxin chimeric can be a Clostridial toxin with
an altered cell binding activity capable of intoxicating a cell
other than a Clostridial toxin target cell, e.g., a cell other than
a motor neuron. Called TVEMPs, these molecules achieve this
intoxication by using a target receptor present on non-Clostridial
toxin target cell. This re-targeted capability is achieved by
replacing a naturally-occurring binding domain of a Clostridial
toxin with a binding domain showing a preferential binding activity
for a non-Clostridial toxin receptor present in a non-Clostridial
toxin target cell. Such modifications to a binding domain result in
a modified toxin that is able to preferentially bind to a
non-Clostridial toxin receptor present on a non-Clostridial toxin
target cell. A Clostridial toxin chimeric with an altered targeting
activity for a non-Clostridial toxin target cell can bind to a
target receptor, translocate into the cytoplasm, and exert its
proteolytic effect on the SNARE complex of the non-Clostridial
toxin target cell. Non-limiting examples of Clostridial toxin
chimeras with an altered targeting activity for a non-Clostridial
toxin target cell are described in, e.g., Keith A. Foster et al.,
Clostridial Toxin Derivatives Able To Modify Peripheral Sensory
Afferent Functions, U.S. Pat. No. 5,989,545; Clifford C. Shone et
al., Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617; Conrad
P. Quinn et al., Methods and Compounds for the Treatment of Mucus
Hypersecretion, U.S. Pat. No. 6,632,440; Lance E. Steward et al.,
Methods and Compositions for the Treatment of Pancreatitis, U.S.
Pat. No. 6,843,998; J. Oliver Dolly et al., Activatable Recombinant
Neurotoxins, U.S. Pat. No. 7,132,259; Stephan Donovan, Clostridial
Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No.
7,244,437; Stephan Donovan, Clostridial Toxin Derivatives and
Methods For Treating Pain, U.S. Pat. No. 7,413,742; Stephan
Donovan, Clostridial Toxin Derivatives and Methods For Treating
Pain, U.S. Pat. No. 7,415,338; Lance E. Steward et al., Multivalent
Clostridial Toxin Derivatives and Methods of Their Use, U.S. Pat.
No. 7,514,088; Keith A. Foster et al., Inhibition of Secretion from
Non-neural Cells, U.S. Patent Publication 2006/0216283; Keith A.
Foster, Fusion Proteins, U.S. Patent Publication 2008/0064092;
Keith A. Foster, Fusion Proteins, U.S. Patent Publication
2009/0035822; Lance E. Steward et al., Multivalent Clostridial
Toxin Derivatives and Methods of Their Use, U.S. Patent Publication
2009/0048431; Keith A. Foster, Non-Cytotoxic Protein Conjugates,
U.S. Patent Publication 2009/0162341; Keith A. Foster et al.,
Re-targeted Toxin Conjugates, International Patent Publication WO
2005/023309; and Lance E. Steward, Modified Clostridial Toxins with
Enhanced Translocation Capabilities and Altered Targeting
Capabilities for Non-Clostridial Toxin Target Cells, International
Patent Application WO 2008/008805; each of which is hereby
incorporated by reference in its entirety.
[0065] Aspects of the present specification provide, in part, a
Clostridial toxin enzymatic domain. As used herein, the term
"Clostridial toxin enzymatic domain" refers to any Clostridial
toxin polypeptide that can execute the enzymatic target
modification step of the intoxication process. Thus, a Clostridial
toxin enzymatic domain specifically targets a Clostridial toxin
substrate and encompasses the proteolytic cleavage of a Clostridial
toxin substrate, such as, e.g., SNARE proteins like a SNAP-25
substrate, a VAMP substrate, and a Syntaxin substrate. Non-limiting
examples of a Clostridial toxin enzymatic domain include, e.g., a
BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1
enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic
domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a
TeNT enzymatic domain, a BaNT enzymatic domain, and a BuNT
enzymatic domain.
[0066] A Clostridial toxin enzymatic domain includes, without
limitation, naturally occurring Clostridial toxin enzymatic domain
variants, such as, e.g., Clostridial toxin enzymatic domain
isoforms and Clostridial toxin enzymatic domain subtypes; and
non-naturally occurring Clostridial toxin enzymatic domain
variants, such as, e.g., conservative Clostridial toxin enzymatic
domain variants, non-conservative Clostridial toxin enzymatic
domain variants, active Clostridial toxin enzymatic domain
fragments thereof, or any combination thereof.
[0067] As used herein, the term "Clostridial toxin enzymatic domain
variant," whether naturally-occurring or non-naturally-occurring,
refers to a Clostridial toxin enzymatic domain that has at least
one amino acid change from the corresponding region of the
disclosed reference sequences (Table 1) and can be described in
percent identity to the corresponding region of that reference
sequence. Unless expressly indicated, Clostridial toxin enzymatic
domain variants useful to practice disclosed embodiments are
variants that execute the enzymatic target modification step of the
intoxication process. As non-limiting examples, a BoNT/A enzymatic
domain variant will have at least one amino acid difference, such
as, e.g., an amino acid substitution, deletion or addition, as
compared to amino acids 1/2-429 of SEQ ID NO: 1; a BoNT/B enzymatic
domain variant will have at least one amino acid difference, such
as, e.g., an amino acid substitution, deletion or addition, as
compared to amino acids 1/2-436 of SEQ ID NO: 6; a BoNT/C1
enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-436 of SEQ ID NO: 11; a
BoNT/D enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-436 of SEQ ID NO: 13; a
BoNT/E enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-411 of SEQ ID NO: 15; a
BoNT/F enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-428 of SEQ ID NO: 18; a
BoNT/G enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-438 of SEQ ID NO: 21; a
TeNT enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-438 of SEQ ID NO: 22; a
BaNT enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-420 of SEQ ID NO: 23; and
a BuNT enzymatic domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 1/2-411 of SEQ ID NO: 24.
[0068] It is recognized by those of skill in the art that within
each serotype of Clostridial toxin there can be naturally occurring
Clostridial toxin enzymatic domain variants that differ somewhat in
their amino acid sequence, and also in the nucleic acids encoding
these proteins. For example, there are presently five BoNT/A
subtypes, BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, and BoNT/A5, with
specific enzymatic domain subtypes showing about 80% to 95% amino
acid identity when compared to the BoNT/A enzymatic domain of SEQ
ID NO: 1. As used herein, the term "naturally occurring Clostridial
toxin enzymatic domain variant" refers to any Clostridial toxin
enzymatic domain produced by a naturally-occurring process,
including, without limitation, Clostridial toxin enzymatic domain
isoforms produced from alternatively-spliced transcripts,
Clostridial toxin enzymatic domain isoforms produced by spontaneous
mutation and Clostridial toxin enzymatic domain subtypes. A
naturally occurring Clostridial toxin enzymatic domain variant can
function in substantially the same manner as the reference
Clostridial toxin enzymatic domain on which the naturally occurring
Clostridial toxin enzymatic domain variant is based, and can be
substituted for the reference Clostridial toxin enzymatic domain in
any aspect of the present specification.
[0069] A non-limiting examples of a naturally occurring Clostridial
toxin enzymatic domain variant is a Clostridial toxin enzymatic
domain isoform such as, e.g., a BoNT/A enzymatic domain isoform, a
BoNT/B enzymatic domain isoform, a BoNT/C1 enzymatic domain
isoform, a BoNT/D enzymatic domain isoform, a BoNT/E enzymatic
domain isoform, a BoNT/F enzymatic domain isoform, a BoNT/G
enzymatic domain isoform, a TeNT enzymatic domain isoform, a BaNT
enzymatic domain isoform, and a BuNT enzymatic domain isoform.
Another non-limiting examples of a naturally occurring Clostridial
toxin enzymatic domain variant is a Clostridial toxin enzymatic
domain subtype such as, e.g., an enzymatic domain from subtype
BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, or BoNT/A5; an enzymatic domain
from subtype BoNT/B1, BoNT/B2, BoNT/Bbv, or BoNT/Bnp; an enzymatic
domain from subtype BoNT/C1-1 or BoNT/C1-2; an enzymatic domain
from subtype BoNT/E1, BoNT/E2 and BoNT/E3; an enzymatic domain from
subtype BoNT/F1, BoNT/F2, or BoNT/F3; and an enzymatic domain from
subtype BuNT-1 or BuNT-2.
[0070] As used herein, the term "non-naturally occurring
Clostridial toxin enzymatic domain variant" refers to any
Clostridial toxin enzymatic domain produced with the aid of human
manipulation, including, without limitation, Clostridial toxin
enzymatic domains produced by genetic engineering using random
mutagenesis or rational design and Clostridial toxin enzymatic
domains produced by chemical synthesis. Non-limiting examples of
non-naturally occurring Clostridial toxin enzymatic domain variants
include, e.g., conservative Clostridial toxin enzymatic domain
variants, non-conservative Clostridial toxin enzymatic domain
variants, Clostridial toxin enzymatic domain chimeric variants, and
active Clostridial toxin enzymatic domain fragments.
[0071] As used herein, the term "conservative Clostridial toxin
enzymatic domain variant" refers to a Clostridial toxin enzymatic
domain that has at least one amino acid substituted by another
amino acid or an amino acid analog that has at least one property
similar to that of the original amino acid from the reference
Clostridial toxin enzymatic domain sequence (Table 1). Examples of
properties include, without limitation, similar size, topography,
charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity, hydrogen-bonding capacity, a
physicochemical property, of the like, or any combination thereof.
A conservative Clostridial toxin enzymatic domain variant can
function in substantially the same manner as the reference
Clostridial toxin enzymatic domain on which the conservative
Clostridial toxin enzymatic domain variant is based, and can be
substituted for the reference Clostridial toxin enzymatic domain in
any aspect of the present specification. Non-limiting examples of a
conservative Clostridial toxin enzymatic domain variant include,
e.g., conservative BoNT/A enzymatic domain variants, conservative
BoNT/B enzymatic domain variants, conservative BoNT/C1 enzymatic
domain variants, conservative BoNT/D enzymatic domain variants,
conservative BoNT/E enzymatic domain variants, conservative BoNT/F
enzymatic domain variants, conservative BoNT/G enzymatic domain
variants, conservative TeNT enzymatic domain variants, conservative
BaNT enzymatic domain variants, and conservative BuNT enzymatic
domain variants.
[0072] As used herein, the term "non-conservative Clostridial toxin
enzymatic domain variant" refers to a Clostridial toxin enzymatic
domain in which 1) at least one amino acid is deleted from the
reference Clostridial toxin enzymatic domain on which the
non-conservative Clostridial toxin enzymatic domain variant is
based; 2) at least one amino acid added to the reference
Clostridial toxin enzymatic domain on which the non-conservative
Clostridial toxin enzymatic domain is based; or 3) at least one
amino acid is substituted by another amino acid or an amino acid
analog that does not share any property similar to that of the
original amino acid from the reference Clostridial toxin enzymatic
domain sequence (Table 1). A non-conservative Clostridial toxin
enzymatic domain variant can function in substantially the same
manner as the reference Clostridial toxin enzymatic domain on which
the non-conservative Clostridial toxin enzymatic domain variant is
based, and can be substituted for the reference Clostridial toxin
enzymatic domain in any aspect of the present specification.
Non-limiting examples of a non-conservative Clostridial toxin
enzymatic domain variant include, e.g., non-conservative BoNT/A
enzymatic domain variants, non-conservative BoNT/B enzymatic domain
variants, non-conservative BoNT/C1 enzymatic domain variants,
non-conservative BoNT/D enzymatic domain variants, non-conservative
BoNT/E enzymatic domain variants, non-conservative BoNT/F enzymatic
domain variants, non-conservative BoNT/G enzymatic domain variants,
and non-conservative TeNT enzymatic domain variants,
non-conservative BaNT enzymatic domain variants, and
non-conservative BuNT enzymatic domain variants.
[0073] As used herein, the term "active Clostridial toxin enzymatic
domain fragment" refers to any of a variety of Clostridial toxin
fragments comprising the enzymatic domain can be useful in aspects
of the present specification with the proviso that these enzymatic
domain fragments can specifically target the core components of the
neurotransmitter release apparatus and thus participate in
executing the overall cellular mechanism whereby a Clostridial
toxin proteolytically cleaves a substrate. The enzymatic domains of
Clostridial toxins are approximately 420-460 amino acids in length
and comprise an enzymatic domain (Table 1). Research has shown that
the entire length of a Clostridial toxin enzymatic domain is not
necessary for the enzymatic activity of the enzymatic domain. As a
non-limiting example, the first eight amino acids of the BoNT/A
enzymatic domain are not required for enzymatic activity. As
another non-limiting example, the first eight amino acids of the
TeNT enzymatic domain are not required for enzymatic activity.
Likewise, the carboxyl-terminus of the enzymatic domain is not
necessary for activity. As a non-limiting example, the last 32
amino acids of the BoNT/A enzymatic domain are not required for
enzymatic activity. As another non-limiting example, the last 31
amino acids of the TeNT enzymatic domain are not required for
enzymatic activity. Thus, aspects of this embodiment include
Clostridial toxin enzymatic domains comprising an enzymatic domain
having a length of, e.g., at least 350, 375, 400, 425, or 450 amino
acids. Other aspects of this embodiment include Clostridial toxin
enzymatic domains comprising an enzymatic domain having a length
of, e.g., at most 350, 375, 400, 425, or 450 amino acids.
[0074] Any of a variety of sequence alignment methods can be used
to determine percent identity of naturally-occurring Clostridial
toxin enzymatic domain variants and non-naturally-occurring
Clostridial toxin enzymatic domain variants, including, without
limitation, global methods, local methods and hybrid methods, such
as, e.g., segment approach methods. Protocols to determine percent
identity are routine procedures within the scope of one skilled in
the art and from the teaching herein.
[0075] Thus, in an embodiment, a modified Clostridial toxin
disclosed in the present specification comprises a Clostridial
toxin enzymatic domain. In an aspect of this embodiment, a
Clostridial toxin enzymatic domain comprises a naturally occurring
Clostridial toxin enzymatic domain variant, such as, e.g., a
Clostridial toxin enzymatic domain isoform or a Clostridial toxin
enzymatic domain subtype. In another aspect of this embodiment, a
Clostridial toxin enzymatic domain comprises a non-naturally
occurring Clostridial toxin enzymatic domain variant, such as,
e.g., a conservative Clostridial toxin enzymatic domain variant, a
non-conservative Clostridial toxin enzymatic domain variant, an
active Clostridial toxin enzymatic domain fragment, or any
combination thereof.
[0076] In another embodiment, a hydrophobic amino acid at one
particular position in the polypeptide chain of the Clostridial
toxin enzymatic domain can be substituted with another hydrophobic
amino acid. Examples of hydrophobic amino acids include, e.g., C,
F, I, L, M, V and W. In another aspect of this embodiment, an
aliphatic amino acid at one particular position in the polypeptide
chain of the Clostridial toxin enzymatic domain can be substituted
with another aliphatic amino acid. Examples of aliphatic amino
acids include, e.g., A, I, L, P, and V. In yet another aspect of
this embodiment, an aromatic amino acid at one particular position
in the polypeptide chain of the Clostridial toxin enzymatic domain
can be substituted with another aromatic amino acid. Examples of
aromatic amino acids include, e.g., F, H, W and Y. In still another
aspect of this embodiment, a stacking amino acid at one particular
position in the polypeptide chain of the Clostridial toxin
enzymatic domain can be substituted with another stacking amino
acid. Examples of stacking amino acids include, e.g., F, H, W and
Y. In a further aspect of this embodiment, a polar amino acid at
one particular position in the polypeptide chain of the Clostridial
toxin enzymatic domain can be substituted with another polar amino
acid. Examples of polar amino acids include, e.g., D, E, K, N, Q,
and R. In a further aspect of this embodiment, a less polar or
indifferent amino acid at one particular position in the
polypeptide chain of the Clostridial toxin enzymatic domain can be
substituted with another less polar or indifferent amino acid.
Examples of less polar or indifferent amino acids include, e.g., A,
H, G, P, S, T, and Y. In a yet further aspect of this embodiment, a
positive charged amino acid at one particular position in the
polypeptide chain of the Clostridial toxin enzymatic domain can be
substituted with another positive charged amino acid. Examples of
positive charged amino acids include, e.g., K, R, and H. In a still
further aspect of this embodiment, a negative charged amino acid at
one particular position in the polypeptide chain of the Clostridial
toxin enzymatic domain can be substituted with another negative
charged amino acid. Examples of negative charged amino acids
include, e.g., D and E. In another aspect of this embodiment, a
small amino acid at one particular position in the polypeptide
chain of the Clostridial toxin enzymatic domain can be substituted
with another small amino acid. Examples of small amino acids
include, e.g., A, D, G, N, P, S, and T. In yet another aspect of
this embodiment, a C-beta branching amino acid at one particular
position in the polypeptide chain of the Clostridial toxin
enzymatic domain can be substituted with another C-beta branching
amino acid. Examples of C-beta branching amino acids include, e.g.,
I, T and V.
[0077] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BoNT/A enzymatic domain. In an aspect of this
embodiment, a BoNT/A enzymatic domain comprises the enzymatic
domains of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
or SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/A
enzymatic domain comprises amino acids 1/2-429 of SEQ ID NO: 1. In
another aspect of this embodiment, a BoNT/A enzymatic domain
comprises a naturally occurring BoNT/A enzymatic domain variant,
such as, e.g., an enzymatic domain from a BoNT/A isoform or an
enzymatic domain from a BoNT/A subtype. In another aspect of this
embodiment, a BoNT/A enzymatic domain comprises a naturally
occurring BoNT/A enzymatic domain variant of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g.,
a BoNT/A isoform enzymatic domain or a BoNT/A subtype enzymatic
domain. In another aspect of this embodiment, a BoNT/A enzymatic
domain comprises amino acids 1/2-429 of a naturally occurring
BoNT/A enzymatic domain variant of SEQ ID NO: 1, such as, e.g., a
BoNT/A isoform enzymatic domain or a BoNT/A subtype enzymatic
domain. In still another aspect of this embodiment, a BoNT/A
enzymatic domain comprises a non-naturally occurring BoNT/A
enzymatic domain variant, such as, e.g., a conservative BoNT/A
enzymatic domain variant, a non-conservative BoNT/A enzymatic
domain variant, an active BoNT/A enzymatic domain fragment, or any
combination thereof. In still another aspect of this embodiment, a
BoNT/A enzymatic domain comprises the enzymatic domain of a
non-naturally occurring BoNT/A enzymatic domain variant of SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5,
such as, e.g., a conservative BoNT/A enzymatic domain variant, a
non-conservative BoNT/A enzymatic domain variant, an active BoNT/A
enzymatic domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/A enzymatic domain
comprises amino acids 1/2-429 of a non-naturally occurring BoNT/A
enzymatic domain variant of SEQ ID NO: 1, such as, e.g., a
conservative BoNT/A enzymatic domain variant, a non-conservative
BoNT/A enzymatic domain variant, an active BoNT/A enzymatic domain
fragment, or any combination thereof.
[0078] In other aspects of this embodiment, a BoNT/A enzymatic
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the enzymatic domain of SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or at
most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at
most 95% to the enzymatic domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ
ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In yet other aspects of
this embodiment, a BoNT/A enzymatic domain comprises a polypeptide
having an amino acid identity of, e.g., at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 1/2-429 of SEQ ID NO: 1; or at most 70%, at most 75%, at most
80%, at most 85%, at most 90%, or at most 95% to amino acids
1/2-429 of SEQ ID NO: 1.
[0079] In other aspects of this embodiment, a BoNT/A enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID
NO: 5. In yet other aspects of this embodiment, a BoNT/A enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
1/2-429 of SEQ ID NO: 1; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-429 of
SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A
enzymatic domain comprises a polypeptide having, e.g., at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID
NO: 5. In further other aspects of this embodiment, a BoNT/A
enzymatic domain comprises a polypeptide having, e.g., at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to amino
acids 1/2-429 of SEQ ID NO: 1; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-429 of
SEQ ID NO: 1.
[0080] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BoNT/B enzymatic domain. In an aspect of this
embodiment, a BoNT/B enzymatic domain comprises the enzymatic
domains of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
or SEQ ID NO: 10. In other aspects of this embodiment, a BoNT/B
enzymatic domain comprises amino acids 1/2-436 of SEQ ID NO: 6. In
another aspect of this embodiment, a BoNT/B enzymatic domain
comprises a naturally occurring BoNT/B enzymatic domain variant,
such as, e.g., an enzymatic domain from a BoNT/B isoform or an
enzymatic domain from a BoNT/B subtype. In another aspect of this
embodiment, a BoNT/B enzymatic domain comprises a naturally
occurring BoNT/B enzymatic domain variant of SEQ ID NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g.,
a BoNT/B isoform enzymatic domain or a BoNT/B subtype enzymatic
domain. In another aspect of this embodiment, a BoNT/B enzymatic
domain comprises amino acids 1/2-436 of a naturally occurring
BoNT/B enzymatic domain variant of SEQ ID NO: 6, such as, e.g., a
BoNT/B isoform enzymatic domain or a BoNT/B subtype enzymatic
domain. In still another aspect of this embodiment, a BoNT/B
enzymatic domain comprises a non-naturally occurring BoNT/B
enzymatic domain variant, such as, e.g., a conservative BoNT/B
enzymatic domain variant, a non-conservative BoNT/B enzymatic
domain variant, an active BoNT/B enzymatic domain fragment, or any
combination thereof. In still another aspect of this embodiment, a
BoNT/B enzymatic domain comprises the enzymatic domain of a
non-naturally occurring BoNT/B enzymatic domain variant of SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10,
such as, e.g., a conservative BoNT/B enzymatic domain variant, a
non-conservative BoNT/B enzymatic domain variant, an active BoNT/B
enzymatic domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/B enzymatic domain
comprises amino acids 1/2-436 of a non-naturally occurring BoNT/B
enzymatic domain variant of SEQ ID NO: 6, such as, e.g., a
conservative BoNT/B enzymatic domain variant, a non-conservative
BoNT/B enzymatic domain variant, an active BoNT/B enzymatic domain
fragment, or any combination thereof.
[0081] In other aspects of this embodiment, a BoNT/B enzymatic
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the enzymatic domain of SEQ ID NO: 6,
SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; or at
most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at
most 95% to the enzymatic domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In yet other aspects of
this embodiment, a BoNT/B enzymatic domain comprises a polypeptide
having an amino acid identity of, e.g., at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 1/2-436 of SEQ ID NO: 6; or at most 70%, at most 75%, at most
80%, at most 85%, at most 90%, or at most 95% to amino acids
1/2-436 of SEQ ID NO: 6.
[0082] In other aspects of this embodiment, a BoNT/B enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID
NO: 10. In yet other aspects of this embodiment, a BoNT/B enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
1/2-436 of SEQ ID NO: 6; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-436 of
SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/B
enzymatic domain comprises a polypeptide having, e.g., at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID
NO: 10. In further other aspects of this embodiment, a BoNT/B
enzymatic domain comprises a polypeptide having, e.g., at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to amino
acids 1/2-436 of SEQ ID NO: 6; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-436 of
SEQ ID NO: 6.
[0083] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BoNT/C1 enzymatic domain. In an aspect of this
embodiment, a BoNT/C1 enzymatic domain comprises the enzymatic
domains of SEQ ID NO: 11 or SEQ ID NO: 12. In other aspects of this
embodiment, a BoNT/C1 enzymatic domain comprises amino acids
1/2-436 of SEQ ID NO: 11. In another aspect of this embodiment, a
BoNT/C1 enzymatic domain comprises a naturally occurring BoNT/C1
enzymatic domain variant, such as, e.g., an enzymatic domain from a
BoNT/C1 isoform or an enzymatic domain from a BoNT/C1 subtype. In
another aspect of this embodiment, a BoNT/C1 enzymatic domain
comprises a naturally occurring BoNT/C1 enzymatic domain variant of
SEQ ID NO: 11 or SEQ ID NO: 12, such as, e.g., a BoNT/C1 isoform
enzymatic domain or a BoNT/C1 subtype enzymatic domain. In another
aspect of this embodiment, a BoNT/C1 enzymatic domain comprises
amino acids 1/2-436 of a naturally occurring BoNT/C1 enzymatic
domain variant of SEQ ID NO: 11, such as, e.g., a BoNT/C1 isoform
enzymatic domain or a BoNT/C1 subtype enzymatic domain. In still
another aspect of this embodiment, a BoNT/C1 enzymatic domain
comprises a non-naturally occurring BoNT/C1 enzymatic domain
variant, such as, e.g., a conservative BoNT/C1 enzymatic domain
variant, a non-conservative BoNT/C1 enzymatic domain variant, an
active BoNT/C1 enzymatic domain fragment, or any combination
thereof. In still another aspect of this embodiment, a BoNT/C1
enzymatic domain comprises the enzymatic domain of a non-naturally
occurring BoNT/C1 enzymatic domain variant of SEQ ID NO: 11 or SEQ
ID NO: 12, such as, e.g., a conservative BoNT/C1 enzymatic domain
variant, a non-conservative BoNT/C1 enzymatic domain variant, an
active BoNT/C1 enzymatic domain fragment, or any combination
thereof. In still another aspect of this embodiment, a BoNT/C1
enzymatic domain comprises amino acids 1/2-436 of a non-naturally
occurring BoNT/C1 enzymatic domain variant of SEQ ID NO: 11, such
as, e.g., a conservative BoNT/C1 enzymatic domain variant, a
non-conservative BoNT/C1 enzymatic domain variant, an active
BoNT/C1 enzymatic domain fragment, or any combination thereof.
[0084] In other aspects of this embodiment, a BoNT/C1 enzymatic
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the enzymatic domain of SEQ ID NO: 11
or SEQ ID NO: 12; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to the enzymatic domain of SEQ ID
NO: 11 or SEQ ID NO: 12. In yet other aspects of this embodiment, a
BoNT/C1 enzymatic domain comprises a polypeptide having an amino
acid identity of, e.g., at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, or at least 95% to amino acids 1/2-436
of SEQ ID NO: 11; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to amino acids 1/2-436 of SEQ ID
NO: 11.
[0085] In other aspects of this embodiment, a BoNT/C1 enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 11 or SEQ ID NO: 12; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid deletions, additions, and/or substitutions relative to
the enzymatic domain of SEQ ID NO: 11 or SEQ ID NO: 12. In yet
other aspects of this embodiment, a BoNT/C1 enzymatic domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
1/2-436 of SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-436 of
SEQ ID NO: 11. In still other aspects of this embodiment, a BoNT/C1
enzymatic domain comprises a polypeptide having, e.g., at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 11 or SEQ ID NO: 12; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 11 or SEQ ID NO: 12. In further
other aspects of this embodiment, a BoNT/C1 enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-436 of
SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-436 of SEQ ID NO: 11.
[0086] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BoNT/D enzymatic domain. In an aspect of this
embodiment, a BoNT/D enzymatic domain comprises the enzymatic
domains of SEQ ID NO: 13 or SEQ ID NO: 14. In other aspects of this
embodiment, a BoNT/D enzymatic domain comprises amino acids 1/2-436
of SEQ ID NO: 13. In another aspect of this embodiment, a BoNT/D
enzymatic domain comprises a naturally occurring BoNT/D enzymatic
domain variant, such as, e.g., an enzymatic domain from a BoNT/D
isoform or an enzymatic domain from a BoNT/D subtype. In another
aspect of this embodiment, a BoNT/D enzymatic domain comprises a
naturally occurring BoNT/D enzymatic domain variant of SEQ ID NO:
13 or SEQ ID NO: 14, such as, e.g., a BoNT/D isoform enzymatic
domain or a BoNT/D subtype enzymatic domain. In another aspect of
this embodiment, a BoNT/D enzymatic domain comprises amino acids
1/2-436 of a naturally occurring BoNT/D enzymatic domain variant of
SEQ ID NO: 13, such as, e.g., a BoNT/D isoform enzymatic domain or
a BoNT/D subtype enzymatic domain. In still another aspect of this
embodiment, a BoNT/D enzymatic domain comprises a non-naturally
occurring BoNT/D enzymatic domain variant, such as, e.g., a
conservative BoNT/D enzymatic domain variant, a non-conservative
BoNT/D enzymatic domain variant, an active BoNT/D enzymatic domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/D enzymatic domain comprises the enzymatic
domain of a non-naturally occurring BoNT/D enzymatic domain variant
of SEQ ID NO: 13 or SEQ ID NO: 14, such as, e.g., a conservative
BoNT/D enzymatic domain variant, a non-conservative BoNT/D
enzymatic domain variant, an active BoNT/D enzymatic domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/D enzymatic domain comprises amino acids
1/2-436 of a non-naturally occurring BoNT/D enzymatic domain
variant of SEQ ID NO: 13, such as, e.g., a conservative BoNT/D
enzymatic domain variant, a non-conservative BoNT/D enzymatic
domain variant, an active BoNT/D enzymatic domain fragment, or any
combination thereof.
[0087] In other aspects of this embodiment, a BoNT/D enzymatic
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the enzymatic domain of SEQ ID NO: 13
or SEQ ID NO: 14; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to the enzymatic domain of SEQ ID
NO: 13 or SEQ ID NO: 14. In yet other aspects of this embodiment, a
BoNT/D enzymatic domain comprises a polypeptide having an amino
acid identity of, e.g., at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, or at least 95% to amino acids 1/2-436
of SEQ ID NO: 13; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to amino acids 1/2-436 of SEQ ID
NO: 13.
[0088] In other aspects of this embodiment, a BoNT/D enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid deletions, additions, and/or substitutions relative to
the enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14. In yet
other aspects of this embodiment, a BoNT/D enzymatic domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
1/2-436 of SEQ ID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-436 of
SEQ ID NO: 13. In still other aspects of this embodiment, a BoNT/D
enzymatic domain comprises a polypeptide having, e.g., at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14. In further
other aspects of this embodiment, a BoNT/D enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-436 of
SEQ ID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-436 of SEQ ID NO: 13.
[0089] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BoNT/E enzymatic domain. In an aspect of this
embodiment, a BoNT/E enzymatic domain comprises the enzymatic
domains of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In other
aspects of this embodiment, a BoNT/E enzymatic domain comprises
amino acids 1/2-411 of SEQ ID NO: 15. In another aspect of this
embodiment, a BoNT/E enzymatic domain comprises a naturally
occurring BoNT/E enzymatic domain variant, such as, e.g., an
enzymatic domain from a BoNT/E isoform or an enzymatic domain from
a BoNT/E subtype. In another aspect of this embodiment, a BoNT/E
enzymatic domain comprises a naturally occurring BoNT/E enzymatic
domain variant of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17,
such as, e.g., a BoNT/E isoform enzymatic domain or a BoNT/E
subtype enzymatic domain. In another aspect of this embodiment, a
BoNT/E enzymatic domain comprises amino acids 1/2-411 of a
naturally occurring BoNT/E enzymatic domain variant of SEQ ID NO:
15, such as, e.g., a BoNT/E isoform enzymatic domain or a BoNT/E
subtype enzymatic domain. In still another aspect of this
embodiment, a BoNT/E enzymatic domain comprises a non-naturally
occurring BoNT/E enzymatic domain variant, such as, e.g., a
conservative BoNT/E enzymatic domain variant, a non-conservative
BoNT/E enzymatic domain variant, an active BoNT/E enzymatic domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/E enzymatic domain comprises the enzymatic
domain of a non-naturally occurring BoNT/E enzymatic domain variant
of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a
conservative BoNT/E enzymatic domain variant, a non-conservative
BoNT/E enzymatic domain variant, an active BoNT/E enzymatic domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/E enzymatic domain comprises amino acids
1/2-411 of a non-naturally occurring BoNT/E enzymatic domain
variant of SEQ ID NO: 15, such as, e.g., a conservative BoNT/E
enzymatic domain variant, a non-conservative BoNT/E enzymatic
domain variant, an active BoNT/E enzymatic domain fragment, or any
combination thereof.
[0090] In other aspects of this embodiment, a BoNT/E enzymatic
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the enzymatic domain of SEQ ID NO:
15, SEQ ID NO: 16, or SEQ ID NO: 17; or at most 70%, at most 75%,
at most 80%, at most 85%, at most 90%, or at most 95% to the
enzymatic domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
In yet other aspects of this embodiment, a BoNT/E enzymatic domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to amino acids 1/2-411 of SEQ ID NO: 15; or at most
70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most
95% to amino acids 1/2-411 of SEQ ID NO: 15.
[0091] In other aspects of this embodiment, a BoNT/E enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17;
or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to the enzymatic domain of SEQ ID NO: 15,
SEQ ID NO: 16, or SEQ ID NO: 17. In yet other aspects of this
embodiment, a BoNT/E enzymatic domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-411 of SEQ ID NO: 15; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-411 of SEQ ID NO: 15. In
still other aspects of this embodiment, a BoNT/E enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or at most 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
In further other aspects of this embodiment, a BoNT/E enzymatic
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
1/2-411 of SEQ ID NO: 15; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 1/2-411 of SEQ ID NO:
15.
[0092] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BoNT/F enzymatic domain. In an aspect of this
embodiment, a BoNT/F enzymatic domain comprises the enzymatic
domains of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In other
aspects of this embodiment, a BoNT/F enzymatic domain comprises
amino acids 1/2-428 of SEQ ID NO: 18. In another aspect of this
embodiment, a BoNT/F enzymatic domain comprises a naturally
occurring BoNT/F enzymatic domain variant, such as, e.g., an
enzymatic domain from a BoNT/F isoform or an enzymatic domain from
a BoNT/F subtype. In another aspect of this embodiment, a BoNT/F
enzymatic domain comprises a naturally occurring BoNT/F enzymatic
domain variant of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20,
such as, e.g., a BoNT/F isoform enzymatic domain or a BoNT/F
subtype enzymatic domain. In another aspect of this embodiment, a
BoNT/F enzymatic domain comprises amino acids 1/2-428 of a
naturally occurring BoNT/F enzymatic domain variant of SEQ ID NO:
18, such as, e.g., a BoNT/F isoform enzymatic domain or a BoNT/F
subtype enzymatic domain. In still another aspect of this
embodiment, a BoNT/F enzymatic domain comprises a non-naturally
occurring BoNT/F enzymatic domain variant, such as, e.g., a
conservative BoNT/F enzymatic domain variant, a non-conservative
BoNT/F enzymatic domain variant, an active BoNT/F enzymatic domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/F enzymatic domain comprises the enzymatic
domain of a non-naturally occurring BoNT/F enzymatic domain variant
of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a
conservative BoNT/F enzymatic domain variant, a non-conservative
BoNT/F enzymatic domain variant, an active BoNT/F enzymatic domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/F enzymatic domain comprises amino acids
1/2-428 of a non-naturally occurring BoNT/F enzymatic domain
variant of SEQ ID NO: 18, such as, e.g., a conservative BoNT/F
enzymatic domain variant, a non-conservative BoNT/F enzymatic
domain variant, an active BoNT/F enzymatic domain fragment, or any
combination thereof.
[0093] In other aspects of this embodiment, a BoNT/F enzymatic
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the enzymatic domain of SEQ ID NO:
18, SEQ ID NO: 19, or SEQ ID NO: 20; or at most 70%, at most 75%,
at most 80%, at most 85%, at most 90%, or at most 95% to the
enzymatic domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.
In yet other aspects of this embodiment, a BoNT/F enzymatic domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to amino acids 1/2-428 of SEQ ID NO: 18; or at most
70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most
95% to amino acids 1/2-428 of SEQ ID NO: 18.
[0094] In other aspects of this embodiment, a BoNT/F enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20;
or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to the enzymatic domain of SEQ ID NO: 18,
SEQ ID NO: 19, or SEQ ID NO: 20. In yet other aspects of this
embodiment, a BoNT/F enzymatic domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-428 of SEQ ID NO: 18; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-428 of SEQ ID NO: 18. In
still other aspects of this embodiment, a BoNT/F enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; or at most 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.
In further other aspects of this embodiment, a BoNT/F enzymatic
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
1/2-428 of SEQ ID NO: 18; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 1/2-428 of SEQ ID NO:
18.
[0095] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BoNT/G enzymatic domain. In an aspect of this
embodiment, a BoNT/G enzymatic domain comprises the enzymatic
domains of SEQ ID NO: 21. In other aspects of this embodiment, a
BoNT/G enzymatic domain comprises amino acids 1/2-4435 of SEQ ID
NO: 21. In another aspect of this embodiment, a BoNT/G enzymatic
domain comprises a naturally occurring BoNT/G enzymatic domain
variant, such as, e.g., an enzymatic domain from a BoNT/G isoform
or an enzymatic domain from a BoNT/G subtype. In another aspect of
this embodiment, a BoNT/G enzymatic domain comprises a naturally
occurring BoNT/G enzymatic domain variant of SEQ ID NO: 21, such
as, e.g., a BoNT/G isoform enzymatic domain or a BoNT/G subtype
enzymatic domain. In another aspect of this embodiment, a BoNT/G
enzymatic domain comprises amino acids 1/2-4435 of a naturally
occurring BoNT/G enzymatic domain variant of SEQ ID NO: 21, such
as, e.g., a BoNT/G isoform enzymatic domain or a BoNT/G subtype
enzymatic domain. In still another aspect of this embodiment, a
BoNT/G enzymatic domain comprises a non-naturally occurring BoNT/G
enzymatic domain variant, such as, e.g., a conservative BoNT/G
enzymatic domain variant, a non-conservative BoNT/G enzymatic
domain variant, an active BoNT/G enzymatic domain fragment, or any
combination thereof. In still another aspect of this embodiment, a
BoNT/G enzymatic domain comprises the enzymatic domain of a
non-naturally occurring BoNT/G enzymatic domain variant of SEQ ID
NO: 21, such as, e.g., a conservative BoNT/G enzymatic domain
variant, a non-conservative BoNT/G enzymatic domain variant, an
active BoNT/G enzymatic domain fragment, or any combination
thereof. In still another aspect of this embodiment, a BoNT/G
enzymatic domain comprises amino acids 1/2-4435 of a non-naturally
occurring BoNT/G enzymatic domain variant of SEQ ID NO: 21, such
as, e.g., a conservative BoNT/G enzymatic domain variant, a
non-conservative BoNT/G enzymatic domain variant, an active BoNT/G
enzymatic domain fragment, or any combination thereof.
[0096] In other aspects of this embodiment, a BoNT/G enzymatic
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the enzymatic domain of SEQ ID NO:
21; or at most 70%, at most 75%, at most 80%, at most 85%, at most
90%, or at most 95% to the enzymatic domain of SEQ ID NO: 21. In
yet other aspects of this embodiment, a BoNT/G enzymatic domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to amino acids 1/2-4435 of SEQ ID NO: 21; or at
most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at
most 95% to amino acids 1/2-4435 of SEQ ID NO: 21.
[0097] In other aspects of this embodiment, a BoNT/G enzymatic
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 21. In yet other aspects of this
embodiment, a BoNT/G enzymatic domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-4435 of SEQ ID NO: 21; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-4435 of SEQ ID NO: 21. In
still other aspects of this embodiment, a BoNT/G enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to the enzymatic domain of SEQ ID NO: 21. In
further other aspects of this embodiment, a BoNT/G enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-4435 of
SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-4435 of SEQ ID NO:
21.
[0098] In another embodiment, a Clostridial toxin enzymatic domain
comprises a TeNT enzymatic domain. In an aspect of this embodiment,
a TeNT enzymatic domain comprises the enzymatic domains of SEQ ID
NO: 22. In other aspects of this embodiment, a TeNT enzymatic
domain comprises amino acids 1/2-438 of SEQ ID NO: 22. In another
aspect of this embodiment, a TeNT enzymatic domain comprises a
naturally occurring TeNT enzymatic domain variant, such as, e.g.,
an enzymatic domain from a TeNT isoform or an enzymatic domain from
a TeNT subtype. In another aspect of this embodiment, a TeNT
enzymatic domain comprises a naturally occurring TeNT enzymatic
domain variant of SEQ ID NO: 22, such as, e.g., a TeNT isoform
enzymatic domain or a TeNT subtype enzymatic domain. In another
aspect of this embodiment, a TeNT enzymatic domain comprises amino
acids 1/2-438 of a naturally occurring TeNT enzymatic domain
variant of SEQ ID NO: 22, such as, e.g., a TeNT isoform enzymatic
domain or a TeNT subtype enzymatic domain. In still another aspect
of this embodiment, a TeNT enzymatic domain comprises a
non-naturally occurring TeNT enzymatic domain variant, such as,
e.g., a conservative TeNT enzymatic domain variant, a
non-conservative TeNT enzymatic domain variant, an active TeNT
enzymatic domain fragment, or any combination thereof. In still
another aspect of this embodiment, a TeNT enzymatic domain
comprises the enzymatic domain of a non-naturally occurring TeNT
enzymatic domain variant of SEQ ID NO: 22, such as, e.g., a
conservative TeNT enzymatic domain variant, a non-conservative TeNT
enzymatic domain variant, an active TeNT enzymatic domain fragment,
or any combination thereof. In still another aspect of this
embodiment, a TeNT enzymatic domain comprises amino acids 1/2-438
of a non-naturally occurring TeNT enzymatic domain variant of SEQ
ID NO: 22, such as, e.g., a conservative TeNT enzymatic domain
variant, a non-conservative TeNT enzymatic domain variant, an
active TeNT enzymatic domain fragment, or any combination
thereof.
[0099] In other aspects of this embodiment, a TeNT enzymatic domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the enzymatic domain of SEQ ID NO: 22; or at
most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at
most 95% to the enzymatic domain of SEQ ID NO: 22. In yet other
aspects of this embodiment, a TeNT enzymatic domain comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to amino acids 1/2-438 of SEQ ID NO: 22; or at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, or at most 95% to
amino acids 1/2-438 of SEQ ID NO: 22.
[0100] In other aspects of this embodiment, a TeNT enzymatic domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 22. In yet other aspects of this
embodiment, a TeNT enzymatic domain comprises a polypeptide having,
e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-438 of SEQ ID NO: 22; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-438 of SEQ ID NO: 22. In
still other aspects of this embodiment, a TeNT enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to the enzymatic domain of SEQ ID NO: 22. In
further other aspects of this embodiment, a TeNT enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-438 of
SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-438 of SEQ ID NO: 22.
[0101] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BaNT enzymatic domain. In an aspect of this embodiment,
a BaNT enzymatic domain comprises the enzymatic domains of SEQ ID
NO: 23. In other aspects of this embodiment, a BaNT enzymatic
domain comprises amino acids 1/2-420 of SEQ ID NO: 23. In another
aspect of this embodiment, a BaNT enzymatic domain comprises a
naturally occurring BaNT enzymatic domain variant, such as, e.g.,
an enzymatic domain from a BaNT isoform or an enzymatic domain from
a BaNT subtype. In another aspect of this embodiment, a BaNT
enzymatic domain comprises a naturally occurring BaNT enzymatic
domain variant of SEQ ID NO: 23, such as, e.g., a BaNT isoform
enzymatic domain or a BaNT subtype enzymatic domain. In another
aspect of this embodiment, a BaNT enzymatic domain comprises amino
acids 1/2-420 of a naturally occurring BaNT enzymatic domain
variant of SEQ ID NO: 23, such as, e.g., a BaNT isoform enzymatic
domain or a BaNT subtype enzymatic domain. In still another aspect
of this embodiment, a BaNT enzymatic domain comprises a
non-naturally occurring BaNT enzymatic domain variant, such as,
e.g., a conservative BaNT enzymatic domain variant, a
non-conservative BaNT enzymatic domain variant, an active BaNT
enzymatic domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BaNT enzymatic domain
comprises the enzymatic domain of a non-naturally occurring BaNT
enzymatic domain variant of SEQ ID NO: 23, such as, e.g., a
conservative BaNT enzymatic domain variant, a non-conservative BaNT
enzymatic domain variant, an active BaNT enzymatic domain fragment,
or any combination thereof. In still another aspect of this
embodiment, a BaNT enzymatic domain comprises amino acids 1/2-420
of a non-naturally occurring BaNT enzymatic domain variant of SEQ
ID NO: 23, such as, e.g., a conservative BaNT enzymatic domain
variant, a non-conservative BaNT enzymatic domain variant, an
active BaNT enzymatic domain fragment, or any combination
thereof.
[0102] In other aspects of this embodiment, a BaNT enzymatic domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the enzymatic domain of SEQ ID NO: 23; or at
most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at
most 95% to the enzymatic domain of SEQ ID NO: 23. In yet other
aspects of this embodiment, a BaNT enzymatic domain comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to amino acids 1/2-420 of SEQ ID NO: 23; or at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, or at most 95% to
amino acids 1/2-420 of SEQ ID NO: 23.
[0103] In other aspects of this embodiment, a BaNT enzymatic domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 23. In yet other aspects of this
embodiment, a BaNT enzymatic domain comprises a polypeptide having,
e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-420 of SEQ ID NO: 23; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-420 of SEQ ID NO: 23. In
still other aspects of this embodiment, a BaNT enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the enzymatic domain of
SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to the enzymatic domain of SEQ ID NO: 23. In
further other aspects of this embodiment, a BaNT enzymatic domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-420 of
SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1/2-420 of SEQ ID NO: 23.
[0104] In another embodiment, a Clostridial toxin enzymatic domain
comprises a BuNT enzymatic domain. In an aspect of this embodiment,
a BuNT enzymatic domain comprises the enzymatic domains of SEQ ID
NO: 24 or SEQ ID NO: 25. In other aspects of this embodiment, a
BuNT enzymatic domain comprises amino acids 1/2-411 of SEQ ID NO:
24. In another aspect of this embodiment, a BuNT enzymatic domain
comprises a naturally occurring BuNT enzymatic domain variant, such
as, e.g., an enzymatic domain from a BuNT isoform or an enzymatic
domain from a BuNT subtype. In another aspect of this embodiment, a
BuNT enzymatic domain comprises a naturally occurring BuNT
enzymatic domain variant of SEQ ID NO: 24 or SEQ ID NO: 25, such
as, e.g., a BuNT isoform enzymatic domain or a BuNT subtype
enzymatic domain. In another aspect of this embodiment, a BuNT
enzymatic domain comprises amino acids 1/2-411 of a naturally
occurring BuNT enzymatic domain variant of SEQ ID NO: 24, such as,
e.g., a BuNT isoform enzymatic domain or a BuNT subtype enzymatic
domain. In still another aspect of this embodiment, a BuNT
enzymatic domain comprises a non-naturally occurring BuNT enzymatic
domain variant, such as, e.g., a conservative BuNT enzymatic domain
variant, a non-conservative BuNT enzymatic domain variant, an
active BuNT enzymatic domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BuNT enzymatic domain
comprises the enzymatic domain of a non-naturally occurring BuNT
enzymatic domain variant of SEQ ID NO: 24 or SEQ ID NO: 25, such
as, e.g., a conservative BuNT enzymatic domain variant, a
non-conservative BuNT enzymatic domain variant, an active BuNT
enzymatic domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BuNT enzymatic domain
comprises amino acids 1/2-411 of a non-naturally occurring BuNT
enzymatic domain variant of SEQ ID NO: 24, such as, e.g., a
conservative BuNT enzymatic domain variant, a non-conservative BuNT
enzymatic domain variant, an active BuNT enzymatic domain fragment,
or any combination thereof.
[0105] In other aspects of this embodiment, a BuNT enzymatic domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the enzymatic domain of SEQ ID NO: 24 or SEQ ID
NO: 25; or at most 70%, at most 75%, at most 80%, at most 85%, at
most 90%, or at most 95% to the enzymatic domain of SEQ ID NO: 24
or SEQ ID NO: 25. In yet other aspects of this embodiment, a BuNT
enzymatic domain comprises a polypeptide having an amino acid
identity of, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% to amino acids 1/2-411 of
SEQ ID NO: 24 or SEQ ID NO: 25; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
1/2-411 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0106] In other aspects of this embodiment, a BuNT enzymatic domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
enzymatic domain of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid deletions, additions, and/or substitutions relative to
the enzymatic domain of SEQ ID NO: 24 OR SEQ ID NO: 25. In yet
other aspects of this embodiment, a BuNT enzymatic domain comprises
a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-411 of
SEQ ID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 1/2-411 of
SEQ ID NO: 24 or SEQ ID NO: 25. In still other aspects of this
embodiment, a BuNT enzymatic domain comprises a polypeptide having,
e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to the enzymatic domain of SEQ ID NO: 24 or
SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to the enzymatic domain of SEQ ID NO: 24 or
SEQ ID NO: 25. In further other aspects of this embodiment, a BuNT
enzymatic domain comprises a polypeptide having, e.g., at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to amino
acids 1/2-411 of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to amino
acids 1/2-411 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0107] The "translocation domain" comprises a portion of a
Clostridial neurotoxin heavy chain having a translocation activity.
By "translocation" is meant the ability to facilitate the transport
of a polypeptide through a vesicular membrane, thereby exposing
some or all of the polypeptide to the cytoplasm. In the various
botulinum neurotoxins translocation is thought to involve an
allosteric conformational change of the heavy chain caused by a
decrease in pH within the endosome. This conformational change
appears to involve and be mediated by the N terminal half of the
heavy chain and to result in the formation of pores in the
vesicular membrane; this change permits the movement of the
proteolytic light chain from within the endosomal vesicle into the
cytoplasm. See e.g., Lacy, et al., Nature Struct. Biol. 5:898-902
(October 1998).
[0108] The amino acid sequence of the translocation-mediating
portion of the botulinum neurotoxin heavy chain is known to those
of skill in the art; additionally, those amino acid residues within
this portion that are known to be essential for conferring the
translocation activity are also known. It would therefore be well
within the ability of one of ordinary skill in the art, for
example, to employ the naturally occurring N-terminal peptide half
of the heavy chain of any of the various Clostridium tetanus or
Clostridium botulinum neurotoxin subtypes as a translocation
domain, or to design an analogous translocation domain by aligning
the primary sequences of the N-terminal halves of the various heavy
chains and selecting a consensus primary translocation sequence
based on conserved amino acid, polarity, steric and hydrophobicity
characteristics between the sequences.
[0109] Aspects of the present specification provide, in part, a
Clostridial toxin translocation domain. As used herein, the term
"Clostridial toxin translocation domain" refers to any Clostridial
toxin polypeptide that can execute the translocation step of the
intoxication process that mediates Clostridial toxin light chain
translocation. Thus, a Clostridial toxin translocation domain
facilitates the movement of a Clostridial toxin light chain across
a membrane and encompasses the movement of a Clostridial toxin
light chain through the membrane an intracellular vesicle into the
cytoplasm of a cell. Non-limiting examples of a Clostridial toxin
translocation domain include, e.g., a BoNT/A translocation domain,
a BoNT/B translocation domain, a BoNT/C1 translocation domain, a
BoNT/D translocation domain, a BoNT/E translocation domain, a
BoNT/F translocation domain, a BoNT/G translocation domain, a TeNT
translocation domain, a BaNT translocation domain, and a BuNT
translocation domain.
[0110] A Clostridial toxin translocation domain includes, without
limitation, naturally occurring Clostridial toxin translocation
domain variants, such as, e.g., Clostridial toxin translocation
domain isoforms and Clostridial toxin translocation domain
subtypes; non-naturally occurring Clostridial toxin translocation
domain variants, such as, e.g., conservative Clostridial toxin
translocation domain variants, non-conservative Clostridial toxin
translocation domain variants, active Clostridial toxin
translocation domain fragments thereof, or any combination
thereof.
[0111] As used herein, the term "Clostridial toxin translocation
domain variant," whether naturally-occurring or
non-naturally-occurring, refers to a Clostridial toxin
translocation domain that has at least one amino acid change from
the corresponding region of the disclosed reference sequences
(Table 1) and can be described in percent identity to the
corresponding region of that reference sequence. Unless expressly
indicated, Clostridial toxin translocation domain variants useful
to practice disclosed embodiments are variants that execute the
translocation step of the intoxication process that mediates
Clostridial toxin light chain translocation. As non-limiting
examples, a BoNT/A translocation domain variant will have at least
one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to amino acids
455-873 of SEQ ID NO: 1; a BoNT/B translocation domain variant will
have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to amino acids
447-860 of SEQ ID NO: 6; a BoNT/C1 translocation domain variant
will have at least one amino acid difference, such as, e.g., an
amino acid substitution, deletion or addition, as compared to amino
acids 454-868 of SEQ ID NO: 11; a BoNT/D translocation domain
variant will have at least one amino acid difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared
to amino acids 451-864 of SEQ ID NO: 13; a BoNT/E translocation
domain variant will have at least one amino acid difference, such
as, e.g., an amino acid substitution, deletion or addition, as
compared to amino acids 427-847 of SEQ ID NO: 15; a BoNT/F
translocation domain variant will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to amino acids 446-865 of SEQ ID NO: 18; a
BoNT/G translocation domain variant will have at least one amino
acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as compared to amino acids 451-865 of SEQ ID
NO: 21; a TeNT translocation domain variant will have at least one
amino acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as compared to amino acids 468-881 of SEQ ID
NO: 22; a BaNT translocation domain variant will have at least one
amino acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as compared to amino acids 436-857 of SEQ ID
NO: 23; and a BuNT translocation domain variant will have at least
one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to amino acids
427-847 of SEQ ID NO: 24.
[0112] It is recognized by those of skill in the art that within
each serotype of Clostridial toxin there can be naturally occurring
Clostridial toxin translocation domain variants that differ
somewhat in their amino acid sequence, and also in the nucleic
acids encoding these proteins. For example, there are presently
five BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, and
BoNT/A5, with specific translocation domain subtypes showing about
85-87% amino acid identity when compared to the BoNT/A
translocation domain subtype of SEQ ID NO: 1. As used herein, the
term "naturally occurring Clostridial toxin translocation domain
variant" refers to any Clostridial toxin translocation domain
produced by a naturally-occurring process, including, without
limitation, Clostridial toxin translocation domain isoforms
produced from alternatively-spliced transcripts, Clostridial toxin
translocation domain isoforms produced by spontaneous mutation and
Clostridial toxin translocation domain subtypes. A naturally
occurring Clostridial toxin translocation domain variant can
function in substantially the same manner as the reference
Clostridial toxin translocation domain on which the naturally
occurring Clostridial toxin translocation domain variant is based,
and can be substituted for the reference Clostridial toxin
translocation domain in any aspect of the present
specification.
[0113] A non-limiting examples of a naturally occurring Clostridial
toxin translocation domain variant is a Clostridial toxin
translocation domain isoform such as, e.g., a BoNT/A translocation
domain isoform, a BoNT/B translocation domain isoform, a BoNT/C1
translocation domain isoform, a BoNT/D translocation domain
isoform, a BoNT/E translocation domain isoform, a BoNT/F
translocation domain isoform, a BoNT/G translocation domain
isoform, a TeNT translocation domain isoform, a BaNT translocation
domain isoform, and a BuNT translocation domain isoform. Another
non-limiting examples of a naturally occurring Clostridial toxin
translocation domain variant is a Clostridial toxin translocation
domain subtype such as, e.g., a translocation domain from subtype
BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, and BoNT/A5; a translocation
domain from subtype BoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/B
nonproteolytic; a translocation domain from subtype BoNT/C1-1 and
BoNT/C1-2; a translocation domain from subtype BoNT/E1, BoNT/E2 and
BoNT/E3; a translocation domain from subtype BoNT/F1, BoNT/F2,
BoNT/F3; and a translocation domain from subtype BuNT-1 and
BuNT-2.
[0114] As used herein, the term "non-naturally occurring
Clostridial toxin translocation domain variant" refers to any
Clostridial toxin translocation domain produced with the aid of
human manipulation, including, without limitation, Clostridial
toxin translocation domains produced by genetic engineering using
random mutagenesis or rational design and Clostridial toxin
translocation domains produced by chemical synthesis. Non-limiting
examples of non-naturally occurring Clostridial toxin translocation
domain variants include, e.g., conservative Clostridial toxin
translocation domain variants, non-conservative Clostridial toxin
translocation domain variants, and active Clostridial toxin
translocation domain fragments.
[0115] As used herein, the term "conservative Clostridial toxin
translocation domain variant" refers to a Clostridial toxin
translocation domain that has at least one amino acid substituted
by another amino acid or an amino acid analog that has at least one
property similar to that of the original amino acid from the
reference Clostridial toxin translocation domain sequence (Table
1). Examples of properties include, without limitation, similar
size, topography, charge, hydrophobicity, hydrophilicity,
lipophilicity, covalent-bonding capacity, hydrogen-bonding
capacity, a physicochemical property, of the like, or any
combination thereof. A conservative Clostridial toxin translocation
domain variant can function in substantially the same manner as the
reference Clostridial toxin translocation domain on which the
conservative Clostridial toxin translocation domain variant is
based, and can be substituted for the reference Clostridial toxin
translocation domain in any aspect of the present specification.
Non-limiting examples of a conservative Clostridial toxin
translocation domain variant include, e.g., conservative BoNT/A
translocation domain variants, conservative BoNT/B translocation
domain variants, conservative BoNT/C1 translocation domain
variants, conservative BoNT/D translocation domain variants,
conservative BoNT/E translocation domain variants, conservative
BoNT/F translocation domain variants, conservative BoNT/G
translocation domain variants, conservative TeNT translocation
domain variants, conservative BaNT translocation domain variants,
and conservative BuNT translocation domain variants.
[0116] As used herein, the term "non-conservative Clostridial toxin
translocation domain variant" refers to a Clostridial toxin
translocation domain in which 1) at least one amino acid is deleted
from the reference Clostridial toxin translocation domain on which
the non-conservative Clostridial toxin translocation domain variant
is based; 2) at least one amino acid added to the reference
Clostridial toxin translocation domain on which the
non-conservative Clostridial toxin translocation domain is based;
or 3) at least one amino acid is substituted by another amino acid
or an amino acid analog that does not share any property similar to
that of the original amino acid from the reference Clostridial
toxin translocation domain sequence (Table 1). A non-conservative
Clostridial toxin translocation domain variant can function in
substantially the same manner as the reference Clostridial toxin
translocation domain on which the non-conservative Clostridial
toxin translocation domain variant is based, and can be substituted
for the reference Clostridial toxin translocation domain in any
aspect of the present specification. Non-limiting examples of a
non-conservative Clostridial toxin translocation domain variant
include, e.g., non-conservative BoNT/A translocation domain
variants, non-conservative BoNT/B translocation domain variants,
non-conservative BoNT/C1 translocation domain variants,
non-conservative BoNT/D translocation domain variants,
non-conservative BoNT/E translocation domain variants,
non-conservative BoNT/F translocation domain variants,
non-conservative BoNT/G translocation domain variants, and
non-conservative TeNT translocation domain variants,
non-conservative BaNT translocation domain variants, and
non-conservative BuNT translocation domain variants.
[0117] As used herein, the term "active Clostridial toxin
translocation domain fragment" refers to any of a variety of
Clostridial toxin fragments comprising the translocation domain can
be useful in aspects of the present specification with the proviso
that these active fragments can facilitate the release of the LC
from intracellular vesicles into the cytoplasm of the target cell
and thus participate in executing the overall cellular mechanism
whereby a Clostridial toxin proteolytically cleaves a substrate.
The translocation domains from the heavy chains of Clostridial
toxins are approximately 410-430 amino acids in length and comprise
a translocation domain (Table 1). Research has shown that the
entire length of a translocation domain from a Clostridial toxin
heavy chain is not necessary for the translocating activity of the
translocation domain. Thus, aspects of this embodiment include a
Clostridial toxin translocation domain having a length of, e.g., at
least 350, 375, 400, or 425 amino acids. Other aspects of this
embodiment include a Clostridial toxin translocation domain having
a length of, e.g., at most 350, 375, 400, or 425 amino acids.
[0118] Any of a variety of sequence alignment methods can be used
to determine percent identity of naturally-occurring Clostridial
toxin translocation domain variants and non-naturally-occurring
Clostridial toxin translocation domain variants, including, without
limitation, global methods, local methods and hybrid methods, such
as, e.g., segment approach methods. Protocols to determine percent
identity are routine procedures within the scope of one skilled in
the art and from the teaching herein.
[0119] Thus, in an embodiment, a modified Clostridial toxin
disclosed in the present specification comprises a Clostridial
toxin translocation domain. In an aspect of this embodiment, a
Clostridial toxin translocation domain comprises a naturally
occurring Clostridial toxin translocation domain variant, such as,
e.g., a Clostridial toxin translocation domain isoform or a
Clostridial toxin translocation domain subtype. In another aspect
of this embodiment, a Clostridial toxin translocation domain
comprises a non-naturally occurring Clostridial toxin translocation
domain variant, such as, e.g., a conservative Clostridial toxin
translocation domain variant, a non-conservative Clostridial toxin
translocation domain variant, an active Clostridial toxin
translocation domain fragment, or any combination thereof.
[0120] In another embodiment, a hydrophobic amino acid at one
particular position in the polypeptide chain of the Clostridial
toxin translocation domain can be substituted with another
hydrophobic amino acid. Examples of hydrophobic amino acids
include, e.g., C, F, I, L, M, V and W. In another aspect of this
embodiment, an aliphatic amino acid at one particular position in
the polypeptide chain of the Clostridial toxin translocation domain
can be substituted with another aliphatic amino acid. Examples of
aliphatic amino acids include, e.g., A, I, L, P, and V. In yet
another aspect of this embodiment, an aromatic amino acid at one
particular position in the polypeptide chain of the Clostridial
toxin translocation domain can be substituted with another aromatic
amino acid. Examples of aromatic amino acids include, e.g., F, H, W
and Y. In still another aspect of this embodiment, a stacking amino
acid at one particular position in the polypeptide chain of the
Clostridial toxin translocation domain can be substituted with
another stacking amino acid. Examples of stacking amino acids
include, e.g., F, H, W and Y. In a further aspect of this
embodiment, a polar amino acid at one particular position in the
polypeptide chain of the Clostridial toxin translocation domain can
be substituted with another polar amino acid. Examples of polar
amino acids include, e.g., D, E, K, N, Q, and R. In a further
aspect of this embodiment, a less polar or indifferent amino acid
at one particular position in the polypeptide chain of the
Clostridial toxin translocation domain can be substituted with
another less polar or indifferent amino acid. Examples of less
polar or indifferent amino acids include, e.g., A, H, G, P, S, T,
and Y. In a yet further aspect of this embodiment, a positive
charged amino acid at one particular position in the polypeptide
chain of the Clostridial toxin translocation domain can be
substituted with another positive charged amino acid. Examples of
positive charged amino acids include, e.g., K, R, and H. In a still
further aspect of this embodiment, a negative charged amino acid at
one particular position in the polypeptide chain of the Clostridial
toxin translocation domain can be substituted with another negative
charged amino acid. Examples of negative charged amino acids
include, e.g., D and E. In another aspect of this embodiment, a
small amino acid at one particular position in the polypeptide
chain of the Clostridial toxin translocation domain can be
substituted with another small amino acid. Examples of small amino
acids include, e.g., A, D, G, N, P, S, and T. In yet another aspect
of this embodiment, a C-beta branching amino acid at one particular
position in the polypeptide chain of the Clostridial toxin
translocation domain can be substituted with another C-beta
branching amino acid. Examples of C-beta branching amino acids
include, e.g., I, T and V.
[0121] In another embodiment, a Clostridial toxin translocation
domain comprises a BoNT/A translocation domain. In an aspect of
this embodiment, a BoNT/A translocation domain comprises the
translocation domains of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, or SEQ ID NO: 5. In other aspects of this embodiment,
a BoNT/A translocation domain comprises amino acids 455-873 of SEQ
ID NO: 1. In another aspect of this embodiment, a BoNT/A
translocation domain comprises a naturally occurring BoNT/A
translocation domain variant, such as, e.g., an translocation
domain from a BoNT/A isoform or an translocation domain from a
BoNT/A subtype. In another aspect of this embodiment, a BoNT/A
translocation domain comprises a naturally occurring BoNT/A
translocation domain variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., a BoNT/A
isoform translocation domain or a BoNT/A subtype translocation
domain. In another aspect of this embodiment, a BoNT/A
translocation domain comprises amino acids 455-873 of a naturally
occurring BoNT/A translocation domain variant of SEQ ID NO: 1, such
as, e.g., a BoNT/A isoform translocation domain or a BoNT/A subtype
translocation domain. In still another aspect of this embodiment, a
BoNT/A translocation domain comprises a non-naturally occurring
BoNT/A translocation domain variant, such as, e.g., a conservative
BoNT/A translocation domain variant, a non-conservative BoNT/A
translocation domain variant, an active BoNT/A translocation domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/A translocation domain comprises the
translocation domain of a non-naturally occurring BoNT/A
translocation domain variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., a conservative
BoNT/A translocation domain variant, a non-conservative BoNT/A
translocation domain variant, an active BoNT/A translocation domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/A translocation domain comprises amino
acids 455-873 of a non-naturally occurring BoNT/A translocation
domain variant of SEQ ID NO: 1, such as, e.g., a conservative
BoNT/A translocation domain variant, a non-conservative BoNT/A
translocation domain variant, an active BoNT/A translocation domain
fragment, or any combination thereof.
[0122] In other aspects of this embodiment, a BoNT/A translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to the translocation domain of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In yet other
aspects of this embodiment, a BoNT/A translocation domain comprises
a polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to amino acids 455-873 of SEQ ID NO: 1; or at most 70%, at most
75%, at most 80%, at most 85%, at most 90%, or at most 95% to amino
acids 455-873 of SEQ ID NO: 1.
[0123] In other aspects of this embodiment, a BoNT/A translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to the translocation
domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
or SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/A
translocation domain comprises a polypeptide having, e.g., at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 455-873 of SEQ ID NO: 1; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 455-873 of SEQ ID NO: 1. In
still other aspects of this embodiment, a BoNT/A translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the translocation
domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
or SEQ ID NO: 5. In further other aspects of this embodiment, a
BoNT/A translocation domain comprises a polypeptide having, e.g.,
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to amino acids 455-873 of SEQ ID NO: 1; or at most 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to amino
acids 455-873 of SEQ ID NO: 1.
[0124] In another embodiment, a Clostridial toxin translocation
domain comprises a BoNT/B translocation domain. In an aspect of
this embodiment, a BoNT/B translocation domain comprises the
translocation domains of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, or SEQ ID NO: 10. In other aspects of this
embodiment, a BoNT/B translocation domain comprises amino acids
447-860 of SEQ ID NO: 6. In another aspect of this embodiment, a
BoNT/B translocation domain comprises a naturally occurring BoNT/B
translocation domain variant, such as, e.g., an translocation
domain from a BoNT/B isoform or an translocation domain from a
BoNT/B subtype. In another aspect of this embodiment, a BoNT/B
translocation domain comprises a naturally occurring BoNT/B
translocation domain variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., a BoNT/B
isoform translocation domain or a BoNT/B subtype translocation
domain. In another aspect of this embodiment, a BoNT/B
translocation domain comprises amino acids 447-860 of a naturally
occurring BoNT/B translocation domain variant of SEQ ID NO: 6, such
as, e.g., a BoNT/B isoform translocation domain or a BoNT/B subtype
translocation domain. In still another aspect of this embodiment, a
BoNT/B translocation domain comprises a non-naturally occurring
BoNT/B translocation domain variant, such as, e.g., a conservative
BoNT/B translocation domain variant, a non-conservative BoNT/B
translocation domain variant, an active BoNT/B translocation domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/B translocation domain comprises the
translocation domain of a non-naturally occurring BoNT/B
translocation domain variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., a
conservative BoNT/B translocation domain variant, a
non-conservative BoNT/B translocation domain variant, an active
BoNT/B translocation domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/B translocation
domain comprises amino acids 447-860 of a non-naturally occurring
BoNT/B translocation domain variant of SEQ ID NO: 6, such as, e.g.,
a conservative BoNT/B translocation domain variant, a
non-conservative BoNT/B translocation domain variant, an active
BoNT/B translocation domain fragment, or any combination
thereof.
[0125] In other aspects of this embodiment, a BoNT/B translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to the translocation domain of SEQ ID NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In yet other
aspects of this embodiment, a BoNT/B translocation domain comprises
a polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to amino acids 447-860 of SEQ ID NO: 6; or at most 70%, at most
75%, at most 80%, at most 85%, at most 90%, or at most 95% to amino
acids 447-860 of SEQ ID NO: 6.
[0126] In other aspects of this embodiment, a BoNT/B translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to the translocation
domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
or SEQ ID NO: 10. In yet other aspects of this embodiment, a BoNT/B
translocation domain comprises a polypeptide having, e.g., at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 447-860 of SEQ ID NO: 6; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 447-860 of SEQ ID NO: 6. In
still other aspects of this embodiment, a BoNT/B translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the translocation
domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
or SEQ ID NO: 10. In further other aspects of this embodiment, a
BoNT/B translocation domain comprises a polypeptide having, e.g.,
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to amino acids 447-860 of SEQ ID NO: 6; or at most 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to amino
acids 447-860 of SEQ ID NO: 6.
[0127] In another embodiment, a Clostridial toxin translocation
domain comprises a BoNT/C1 translocation domain. In an aspect of
this embodiment, a BoNT/C1 translocation domain comprises the
translocation domains of SEQ ID NO: 11 or SEQ ID NO: 12. In other
aspects of this embodiment, a BoNT/C1 translocation domain
comprises amino acids 454-868 of SEQ ID NO: 11. In another aspect
of this embodiment, a BoNT/C1 translocation domain comprises a
naturally occurring BoNT/C1 translocation domain variant, such as,
e.g., an translocation domain from a BoNT/C1 isoform or an
translocation domain from a BoNT/C1 subtype. In another aspect of
this embodiment, a BoNT/C1 translocation domain comprises a
naturally occurring BoNT/C1 translocation domain variant of SEQ ID
NO: 11 or SEQ ID NO: 12, such as, e.g., a BoNT/C1 isoform
translocation domain or a BoNT/C1 subtype translocation domain. In
another aspect of this embodiment, a BoNT/C1 translocation domain
comprises amino acids 454-868 of a naturally occurring BoNT/C1
translocation domain variant of SEQ ID NO: 11, such as, e.g., a
BoNT/C1 isoform translocation domain or a BoNT/C1 subtype
translocation domain. In still another aspect of this embodiment, a
BoNT/C1 translocation domain comprises a non-naturally occurring
BoNT/C1 translocation domain variant, such as, e.g., a conservative
BoNT/C1 translocation domain variant, a non-conservative BoNT/C1
translocation domain variant, an active BoNT/C1 translocation
domain fragment, or any combination thereof. In still another
aspect of this embodiment, a BoNT/C1 translocation domain comprises
the translocation domain of a non-naturally occurring BoNT/C1
translocation domain variant of SEQ ID NO: 11 or SEQ ID NO: 12,
such as, e.g., a conservative BoNT/C1 translocation domain variant,
a non-conservative BoNT/C1 translocation domain variant, an active
BoNT/C1 translocation domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/C1 translocation
domain comprises amino acids 454-868 of a non-naturally occurring
BoNT/C1 translocation domain variant of SEQ ID NO: 11, such as,
e.g., a conservative BoNT/C1 translocation domain variant, a
non-conservative BoNT/C1 translocation domain variant, an active
BoNT/C1 translocation domain fragment, or any combination
thereof.
[0128] In other aspects of this embodiment, a BoNT/C1 translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 11 or SEQ ID NO: 12; or at most 70%, at most 75%, at most 80%,
at most 85%, at most 90%, or at most 95% to the translocation
domain of SEQ ID NO: 11 or SEQ ID NO: 12. In yet other aspects of
this embodiment, a BoNT/C1 translocation domain comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to amino acids 454-868 of SEQ ID NO: 11; or at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, or at most 95% to
amino acids 454-868 of SEQ ID NO: 11.
[0129] In other aspects of this embodiment, a BoNT/C1 translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 11 or SEQ ID NO: 12; or at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO: 11
or SEQ ID NO: 12. In yet other aspects of this embodiment, a
BoNT/C1 translocation domain comprises a polypeptide having, e.g.,
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 454-868 of SEQ ID NO: 11; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 454-868 of SEQ ID NO: 11. In
still other aspects of this embodiment, a BoNT/C1 translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 11 or SEQ ID NO: 12; or at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous
amino acid deletions, additions, and/or substitutions relative to
the translocation domain of SEQ ID NO: 11 or SEQ ID NO: 12. In
further other aspects of this embodiment, a BoNT/C1 translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
454-868 of SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 454-868 of SEQ ID NO:
11.
[0130] In another embodiment, a Clostridial toxin translocation
domain comprises a BoNT/D translocation domain. In an aspect of
this embodiment, a BoNT/D translocation domain comprises the
translocation domains of SEQ ID NO: 13 or SEQ ID NO: 14. In other
aspects of this embodiment, a BoNT/D translocation domain comprises
amino acids 451-864 of SEQ ID NO: 13. In another aspect of this
embodiment, a BoNT/D translocation domain comprises a naturally
occurring BoNT/D translocation domain variant, such as, e.g., an
translocation domain from a BoNT/D isoform or an translocation
domain from a BoNT/D subtype. In another aspect of this embodiment,
a BoNT/D translocation domain comprises a naturally occurring
BoNT/D translocation domain variant of SEQ ID NO: 13 or SEQ ID NO:
14, such as, e.g., a BoNT/D isoform translocation domain or a
BoNT/D subtype translocation domain. In another aspect of this
embodiment, a BoNT/D translocation domain comprises amino acids
451-864 of a naturally occurring BoNT/D translocation domain
variant of SEQ ID NO: 13, such as, e.g., a BoNT/D isoform
translocation domain or a BoNT/D subtype translocation domain. In
still another aspect of this embodiment, a BoNT/D translocation
domain comprises a non-naturally occurring BoNT/D translocation
domain variant, such as, e.g., a conservative BoNT/D translocation
domain variant, a non-conservative BoNT/D translocation domain
variant, an active BoNT/D translocation domain fragment, or any
combination thereof. In still another aspect of this embodiment, a
BoNT/D translocation domain comprises the translocation domain of a
non-naturally occurring BoNT/D translocation domain variant of SEQ
ID NO: 13 or SEQ ID NO: 14, such as, e.g., a conservative BoNT/D
translocation domain variant, a non-conservative BoNT/D
translocation domain variant, an active BoNT/D translocation domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/D translocation domain comprises amino
acids 451-864 of a non-naturally occurring BoNT/D translocation
domain variant of SEQ ID NO: 13, such as, e.g., a conservative
BoNT/D translocation domain variant, a non-conservative BoNT/D
translocation domain variant, an active BoNT/D translocation domain
fragment, or any combination thereof.
[0131] In other aspects of this embodiment, a BoNT/D translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 13 or SEQ ID NO: 14; or at most 70%, at most 75%, at most 80%,
at most 85%, at most 90%, or at most 95% to the translocation
domain of SEQ ID NO: 13 or SEQ ID NO: 14. In yet other aspects of
this embodiment, a BoNT/D translocation domain comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to amino acids 451-864 of SEQ ID NO: 13; or at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, or at most 95% to
amino acids 451-864 of SEQ ID NO: 13.
[0132] In other aspects of this embodiment, a BoNT/D translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO: 13
or SEQ ID NO: 14. In yet other aspects of this embodiment, a BoNT/D
translocation domain comprises a polypeptide having, e.g., at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 451-864 of SEQ ID NO: 13; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 451-864 of SEQ ID NO: 13. In
still other aspects of this embodiment, a BoNT/D translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous
amino acid deletions, additions, and/or substitutions relative to
the translocation domain of SEQ ID NO: 13 or SEQ ID NO: 14. In
further other aspects of this embodiment, a BoNT/D translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
451-864 of SEQ ID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 451-864 of SEQ ID NO:
13.
[0133] In another embodiment, a Clostridial toxin translocation
domain comprises a BoNT/E translocation domain. In an aspect of
this embodiment, a BoNT/E translocation domain comprises the
translocation domains of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID
NO: 17. In other aspects of this embodiment, a BoNT/E translocation
domain comprises amino acids 427-847 of SEQ ID NO: 15. In another
aspect of this embodiment, a BoNT/E translocation domain comprises
a naturally occurring BoNT/E translocation domain variant, such as,
e.g., an translocation domain from a BoNT/E isoform or an
translocation domain from a BoNT/E subtype. In another aspect of
this embodiment, a BoNT/E translocation domain comprises a
naturally occurring BoNT/E translocation domain variant of SEQ ID
NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a BoNT/E
isoform translocation domain or a BoNT/E subtype translocation
domain. In another aspect of this embodiment, a BoNT/E
translocation domain comprises amino acids 427-847 of a naturally
occurring BoNT/E translocation domain variant of SEQ ID NO: 15,
such as, e.g., a BoNT/E isoform translocation domain or a BoNT/E
subtype translocation domain. In still another aspect of this
embodiment, a BoNT/E translocation domain comprises a non-naturally
occurring BoNT/E translocation domain variant, such as, e.g., a
conservative BoNT/E translocation domain variant, a
non-conservative BoNT/E translocation domain variant, an active
BoNT/E translocation domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/E translocation
domain comprises the translocation domain of a non-naturally
occurring BoNT/E translocation domain variant of SEQ ID NO: 15, SEQ
ID NO: 16, or SEQ ID NO: 17, such as, e.g., a conservative BoNT/E
translocation domain variant, a non-conservative BoNT/E
translocation domain variant, an active BoNT/E translocation domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/E translocation domain comprises amino
acids 427-847 of a non-naturally occurring BoNT/E translocation
domain variant of SEQ ID NO: 15, such as, e.g., a conservative
BoNT/E translocation domain variant, a non-conservative BoNT/E
translocation domain variant, an active BoNT/E translocation domain
fragment, or any combination thereof.
[0134] In other aspects of this embodiment, a BoNT/E translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or at most 70%, at most
75%, at most 80%, at most 85%, at most 90%, or at most 95% to the
translocation domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:
17. In yet other aspects of this embodiment, a BoNT/E translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to amino acids 427-847 of SEQ ID NO: 15;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to amino acids 427-847 of SEQ ID NO: 15.
[0135] In other aspects of this embodiment, a BoNT/E translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:
17; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO:
15, SEQ ID NO: 16, or SEQ ID NO: 17. In yet other aspects of this
embodiment, a BoNT/E translocation domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 427-847 of SEQ ID NO: 15; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 427-847 of SEQ ID NO: 15. In
still other aspects of this embodiment, a BoNT/E translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:
17; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO:
15, SEQ ID NO: 16, or SEQ ID NO: 17. In further other aspects of
this embodiment, a BoNT/E translocation domain comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 427-847 of SEQ ID NO:
15; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 427-847 of SEQ ID NO: 15.
[0136] In another embodiment, a Clostridial toxin translocation
domain comprises a BoNT/F translocation domain. In an aspect of
this embodiment, a BoNT/F translocation domain comprises the
translocation domains of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID
NO: 20. In other aspects of this embodiment, a BoNT/F translocation
domain comprises amino acids 446-865 of SEQ ID NO: 18. In another
aspect of this embodiment, a BoNT/F translocation domain comprises
a naturally occurring BoNT/F translocation domain variant, such as,
e.g., an translocation domain from a BoNT/F isoform or an
translocation domain from a BoNT/F subtype. In another aspect of
this embodiment, a BoNT/F translocation domain comprises a
naturally occurring BoNT/F translocation domain variant of SEQ ID
NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a BoNT/F
isoform translocation domain or a BoNT/F subtype translocation
domain. In another aspect of this embodiment, a BoNT/F
translocation domain comprises amino acids 446-865 of a naturally
occurring BoNT/F translocation domain variant of SEQ ID NO: 18,
such as, e.g., a BoNT/F isoform translocation domain or a BoNT/F
subtype translocation domain. In still another aspect of this
embodiment, a BoNT/F translocation domain comprises a non-naturally
occurring BoNT/F translocation domain variant, such as, e.g., a
conservative BoNT/F translocation domain variant, a
non-conservative BoNT/F translocation domain variant, an active
BoNT/F translocation domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/F translocation
domain comprises the translocation domain of a non-naturally
occurring BoNT/F translocation domain variant of SEQ ID NO: 18, SEQ
ID NO: 19, or SEQ ID NO: 20, such as, e.g., a conservative BoNT/F
translocation domain variant, a non-conservative BoNT/F
translocation domain variant, an active BoNT/F translocation domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/F translocation domain comprises amino
acids 446-865 of a non-naturally occurring BoNT/F translocation
domain variant of SEQ ID NO: 18, such as, e.g., a conservative
BoNT/F translocation domain variant, a non-conservative BoNT/F
translocation domain variant, an active BoNT/F translocation domain
fragment, or any combination thereof.
[0137] In other aspects of this embodiment, a BoNT/F translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; or at most 70%, at most
75%, at most 80%, at most 85%, at most 90%, or at most 95% to the
translocation domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:
20. In yet other aspects of this embodiment, a BoNT/F translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to amino acids 446-865 of SEQ ID NO: 18;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to amino acids 446-865 of SEQ ID NO: 18.
[0138] In other aspects of this embodiment, a BoNT/F translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:
20; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO:
18, SEQ ID NO: 19, or SEQ ID NO: 20. In yet other aspects of this
embodiment, a BoNT/F translocation domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 446-865 of SEQ ID NO: 18; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 446-865 of SEQ ID NO: 18. In
still other aspects of this embodiment, a BoNT/F translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:
20; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO:
18, SEQ ID NO: 19, or SEQ ID NO: 20. In further other aspects of
this embodiment, a BoNT/F translocation domain comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 446-865 of SEQ ID NO:
18; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 446-865 of SEQ ID NO: 18.
[0139] In another embodiment, a Clostridial toxin translocation
domain comprises a BoNT/G translocation domain. In an aspect of
this embodiment, a BoNT/G translocation domain comprises the
translocation domains of SEQ ID NO: 21. In other aspects of this
embodiment, a BoNT/G translocation domain comprises amino acids
451-865 of SEQ ID NO: 21. In another aspect of this embodiment, a
BoNT/G translocation domain comprises a naturally occurring BoNT/G
translocation domain variant, such as, e.g., an translocation
domain from a BoNT/G isoform or an translocation domain from a
BoNT/G subtype. In another aspect of this embodiment, a BoNT/G
translocation domain comprises a naturally occurring BoNT/G
translocation domain variant of SEQ ID NO: 21, such as, e.g., a
BoNT/G isoform translocation domain or a BoNT/G subtype
translocation domain. In another aspect of this embodiment, a
BoNT/G translocation domain comprises amino acids 451-865 of a
naturally occurring BoNT/G translocation domain variant of SEQ ID
NO: 21, such as, e.g., a BoNT/G isoform translocation domain or a
BoNT/G subtype translocation domain. In still another aspect of
this embodiment, a BoNT/G translocation domain comprises a
non-naturally occurring BoNT/G translocation domain variant, such
as, e.g., a conservative BoNT/G translocation domain variant, a
non-conservative BoNT/G translocation domain variant, an active
BoNT/G translocation domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/G translocation
domain comprises the translocation domain of a non-naturally
occurring BoNT/G translocation domain variant of SEQ ID NO: 21,
such as, e.g., a conservative BoNT/G translocation domain variant,
a non-conservative BoNT/G translocation domain variant, an active
BoNT/G translocation domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/G translocation
domain comprises amino acids 451-865 of a non-naturally occurring
BoNT/G translocation domain variant of SEQ ID NO: 21, such as,
e.g., a conservative BoNT/G translocation domain variant, a
non-conservative BoNT/G translocation domain variant, an active
BoNT/G translocation domain fragment, or any combination
thereof.
[0140] In other aspects of this embodiment, a BoNT/G translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 21; or at most 70%, at most 75%, at most 80%, at most 85%, at
most 90%, or at most 95% to the translocation domain of SEQ ID NO:
21. In yet other aspects of this embodiment, a BoNT/G translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to amino acids 451-865 of SEQ ID NO: 21;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to amino acids 451-865 of SEQ ID NO: 21.
[0141] In other aspects of this embodiment, a BoNT/G translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 21. In yet other aspects of this
embodiment, a BoNT/G translocation domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 451-865 of SEQ ID NO: 21; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 451-865 of SEQ ID NO: 21. In
still other aspects of this embodiment, a BoNT/G translocation
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 21. In further other aspects of
this embodiment, a BoNT/G translocation domain comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 451-865 of SEQ ID NO:
21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 451-865 of SEQ ID NO: 21.
[0142] In another embodiment, a Clostridial toxin translocation
domain comprises a TeNT translocation domain. In an aspect of this
embodiment, a TeNT translocation domain comprises the translocation
domains of SEQ ID NO: 22. In other aspects of this embodiment, a
TeNT translocation domain comprises amino acids 468-881 of SEQ ID
NO: 22. In another aspect of this embodiment, a TeNT translocation
domain comprises a naturally occurring TeNT translocation domain
variant, such as, e.g., an translocation domain from a TeNT isoform
or an translocation domain from a TeNT subtype. In another aspect
of this embodiment, a TeNT translocation domain comprises a
naturally occurring TeNT translocation domain variant of SEQ ID NO:
22, such as, e.g., a TeNT isoform translocation domain or a TeNT
subtype translocation domain. In another aspect of this embodiment,
a TeNT translocation domain comprises amino acids 468-881 of a
naturally occurring TeNT translocation domain variant of SEQ ID NO:
22, such as, e.g., a TeNT isoform translocation domain or a TeNT
subtype translocation domain. In still another aspect of this
embodiment, a TeNT translocation domain comprises a non-naturally
occurring TeNT translocation domain variant, such as, e.g., a
conservative TeNT translocation domain variant, a non-conservative
TeNT translocation domain variant, an active TeNT translocation
domain fragment, or any combination thereof. In still another
aspect of this embodiment, a TeNT translocation domain comprises
the translocation domain of a non-naturally occurring TeNT
translocation domain variant of SEQ ID NO: 22, such as, e.g., a
conservative TeNT translocation domain variant, a non-conservative
TeNT translocation domain variant, an active TeNT translocation
domain fragment, or any combination thereof. In still another
aspect of this embodiment, a TeNT translocation domain comprises
amino acids 468-881 of a non-naturally occurring TeNT translocation
domain variant of SEQ ID NO: 22, such as, e.g., a conservative TeNT
translocation domain variant, a non-conservative TeNT translocation
domain variant, an active TeNT translocation domain fragment, or
any combination thereof.
[0143] In other aspects of this embodiment, a TeNT translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 22; or at most 70%, at most 75%, at most 80%, at most 85%, at
most 90%, or at most 95% to the translocation domain of SEQ ID NO:
22. In yet other aspects of this embodiment, a TeNT translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to amino acids 468-881 of SEQ ID NO: 22;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to amino acids 468-881 of SEQ ID NO: 22.
[0144] In other aspects of this embodiment, a TeNT translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 22. In yet other aspects of this
embodiment, a TeNT translocation domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 468-881 of SEQ ID NO: 22; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 468-881 of SEQ ID NO: 22. In
still other aspects of this embodiment, a TeNT translocation domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the translocation
domain of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to the translocation domain of SEQ ID
NO: 22. In further other aspects of this embodiment, a TeNT
translocation domain comprises a polypeptide having, e.g., at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous
amino acid deletions, additions, and/or substitutions relative to
amino acids 468-881 of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
468-881 of SEQ ID NO: 22.
[0145] In another embodiment, a Clostridial toxin translocation
domain comprises a BaNT translocation domain. In an aspect of this
embodiment, a BaNT translocation domain comprises the translocation
domains of SEQ ID NO: 23. In other aspects of this embodiment, a
BaNT translocation domain comprises amino acids 436-857 of SEQ ID
NO: 23. In another aspect of this embodiment, a BaNT translocation
domain comprises a naturally occurring BaNT translocation domain
variant, such as, e.g., an translocation domain from a BaNT isoform
or an translocation domain from a BaNT subtype. In another aspect
of this embodiment, a BaNT translocation domain comprises a
naturally occurring BaNT translocation domain variant of SEQ ID NO:
23, such as, e.g., a BaNT isoform translocation domain or a BaNT
subtype translocation domain. In another aspect of this embodiment,
a BaNT translocation domain comprises amino acids 436-857 of a
naturally occurring BaNT translocation domain variant of SEQ ID NO:
23, such as, e.g., a BaNT isoform translocation domain or a BaNT
subtype translocation domain. In still another aspect of this
embodiment, a BaNT translocation domain comprises a non-naturally
occurring BaNT translocation domain variant, such as, e.g., a
conservative BaNT translocation domain variant, a non-conservative
BaNT translocation domain variant, an active BaNT translocation
domain fragment, or any combination thereof. In still another
aspect of this embodiment, a BaNT translocation domain comprises
the translocation domain of a non-naturally occurring BaNT
translocation domain variant of SEQ ID NO: 23, such as, e.g., a
conservative BaNT translocation domain variant, a non-conservative
BaNT translocation domain variant, an active BaNT translocation
domain fragment, or any combination thereof. In still another
aspect of this embodiment, a BaNT translocation domain comprises
amino acids 436-857 of a non-naturally occurring BaNT translocation
domain variant of SEQ ID NO: 23, such as, e.g., a conservative BaNT
translocation domain variant, a non-conservative BaNT translocation
domain variant, an active BaNT translocation domain fragment, or
any combination thereof.
[0146] In other aspects of this embodiment, a BaNT translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 23; or at most 70%, at most 75%, at most 80%, at most 85%, at
most 90%, or at most 95% to the translocation domain of SEQ ID NO:
23. In yet other aspects of this embodiment, a BaNT translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to amino acids 436-857 of SEQ ID NO: 23;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to amino acids 436-857 of SEQ ID NO: 23.
[0147] In other aspects of this embodiment, a BaNT translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 23. In yet other aspects of this
embodiment, a BaNT translocation domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 436-857 of SEQ ID NO: 23; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 436-857 of SEQ ID NO: 23. In
still other aspects of this embodiment, a BaNT translocation domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the translocation
domain of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to the translocation domain of SEQ ID
NO: 23. In further other aspects of this embodiment, a BaNT
translocation domain comprises a polypeptide having, e.g., at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous
amino acid deletions, additions, and/or substitutions relative to
amino acids 436-857 of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
436-857 of SEQ ID NO: 23.
[0148] In another embodiment, a Clostridial toxin translocation
domain comprises a BuNT translocation domain. In an aspect of this
embodiment, a BuNT translocation domain comprises the translocation
domains of SEQ ID NO: 24 or SEQ ID NO: 25. In other aspects of this
embodiment, a BuNT translocation domain comprises amino acids
427-847 of SEQ ID NO: 24. In another aspect of this embodiment, a
BuNT translocation domain comprises a naturally occurring BuNT
translocation domain variant, such as, e.g., an translocation
domain from a BuNT isoform or an translocation domain from a BuNT
subtype. In another aspect of this embodiment, a BuNT translocation
domain comprises a naturally occurring BuNT translocation domain
variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g., a BuNT
isoform translocation domain or a BuNT subtype translocation
domain. In another aspect of this embodiment, a BuNT translocation
domain comprises amino acids 427-847 of a naturally occurring BuNT
translocation domain variant of SEQ ID NO: 24, such as, e.g., a
BuNT isoform translocation domain or a BuNT subtype translocation
domain. In still another aspect of this embodiment, a BuNT
translocation domain comprises a non-naturally occurring BuNT
translocation domain variant, such as, e.g., a conservative BuNT
translocation domain variant, a non-conservative BuNT translocation
domain variant, an active BuNT translocation domain fragment, or
any combination thereof. In still another aspect of this
embodiment, a BuNT translocation domain comprises the translocation
domain of a non-naturally occurring BuNT translocation domain
variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g., a
conservative BuNT translocation domain variant, a non-conservative
BuNT translocation domain variant, an active BuNT translocation
domain fragment, or any combination thereof. In still another
aspect of this embodiment, a BuNT translocation domain comprises
amino acids 427-847 of a non-naturally occurring BuNT translocation
domain variant of SEQ ID NO: 24, such as, e.g., a conservative BuNT
translocation domain variant, a non-conservative BuNT translocation
domain variant, an active BuNT translocation domain fragment, or
any combination thereof.
[0149] In other aspects of this embodiment, a BuNT translocation
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the translocation domain of SEQ ID
NO: 24 or SEQ ID NO: 25; or at most 70%, at most 75%, at most 80%,
at most 85%, at most 90%, or at most 95% to the translocation
domain of SEQ ID NO: 24 or SEQ ID NO: 25. In yet other aspects of
this embodiment, a BuNT translocation domain comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO: 25; or at
most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at
most 95% to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO:
25.
[0150] In other aspects of this embodiment, a BuNT translocation
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the
translocation domain of SEQ ID NO: 24 or SEQ ID NO: 25; or at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO: 24
OR SEQ ID NO: 25. In yet other aspects of this embodiment, a BuNT
translocation domain comprises a polypeptide having, e.g., at most
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 427-847 of SEQ ID NO: 24 or
SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 non-contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 427-847 of SEQ ID NO:
24 or SEQ ID NO: 25. In still other aspects of this embodiment, a
BuNT translocation domain comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to the translocation domain of SEQ ID NO: 24 or SEQ ID NO:
25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to the translocation domain of SEQ ID NO: 24
or SEQ ID NO: 25. In further other aspects of this embodiment, a
BuNT translocation domain comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO: 25;
or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO:
25.
[0151] Aspects of the present specification provide, in part, a
binding domain. As used herein, the term "binding domain" is
synonymous with "ligand" or "targeting moiety" and refers to any
molecule that can preferentially interact with another molecule
present on the surface of a cell under physiological conditions.
The cell surface molecule may comprise a polypeptide, a
polysaccharide, a lipid, or may have structural characteristics of
more than one of these. As used herein, the term "preferentially
interacts" refers to molecule is able to bind its target receptor
under physiological conditions, or in vitro conditions
substantially approximating physiological conditions, to a
statistically significantly greater degree relative to other,
non-target receptor. With reference to a Clostridial toxin binding
domain disclosed in the present specification, there is a
discriminatory binding of the Clostridial toxin binding domain to
its cognate receptor relative to other receptors. With reference to
a non-Clostridial toxin binding domain disclosed in the present
specification, there is a discriminatory binding of the
non-Clostridial toxin binding domain to it cognate receptor
relative to other receptors.
[0152] Thus, in an embodiment, a binding domain that selectively
binds a target receptor has a dissociation equilibrium constant
(K.sub.D) that is greater for the target receptor relative to a
non-target receptor by, e.g., at least one-fold, at least two-fold,
at least three-fold, at least four fold, at least five-fold, at
least 10 fold, at least 50 fold, at least 100 fold, at least 1000,
at least 10,000, or at least 100,000 fold.
[0153] Aspects of the present specification provide, in part, a
Clostridial toxin binding domain. As used herein, the term
"Clostridial toxin binding domain" refers to any Clostridial toxin
polypeptide that can execute the binding step of the intoxication
process that initiates the overall internalization mechanism
whereby the modified Clostridial toxin disclosed in the present
specification intoxicates a target cell. Non-limiting examples of a
Clostridial toxin binding domain include, e.g., a BoNT/A binding
domain, a BoNT/B binding domain, a BoNT/C1 binding domain, a BoNT/D
binding domain, a BoNT/E binding domain, a BoNT/F binding domain, a
BoNT/G binding domain, a TeNT binding domain, a BaNT binding
domain, and a BuNT binding domain. Other non-limiting examples of a
Clostridial toxin binding domain include, e.g., amino acids
874-1296 of SEQ ID NO: 1, amino acids 861-1291 of SEQ ID NO: 2,
amino acids 869-1291 of SEQ ID NO: 3, amino acids 865-1291 of SEQ
ID NO: 4, amino acids 848-1252 of SEQ ID NO: 5, amino acids
866-1274 of SEQ ID NO: 6, amino acids 866-1297 of SEQ ID NO: 7,
amino acids 882-1315 of SEQ ID NO: 8, amino acids 858-1268 of SEQ
ID NO: 9, and amino acids 848-1251 of SEQ ID NO: 10.
[0154] A Clostridial toxin binding domain includes, without
limitation, naturally occurring Clostridial toxin binding domain
variants, such as, e.g., Clostridial toxin binding domain isoforms
and Clostridial toxin binding domain subtypes; non-naturally
occurring Clostridial toxin binding domain variants, such as, e.g.,
conservative Clostridial toxin binding domain variants,
non-conservative Clostridial toxin binding domain variants, active
Clostridial toxin binding domain fragments thereof, or any
combination thereof.
[0155] As used herein, the term "Clostridial toxin binding domain
variant," whether naturally-occurring or non-naturally-occurring,
refers to a Clostridial toxin binding domain that has at least one
amino acid change from the corresponding region of the disclosed
reference sequences (Table 1) and can be described in percent
identity to the corresponding region of that reference sequence.
Unless expressly indicated, Clostridial toxin binding domain
variants useful to practice disclosed embodiments are variants that
execute the translocation step of the intoxication process that
mediates Clostridial toxin light chain translocation. As
non-limiting examples, a BoNT/A binding domain variant will have at
least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to amino acids
874-1296 of SEQ ID NO: 1; a BoNT/B binding domain variant will have
at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to amino acids
861-1291 of SEQ ID NO: 6; a BoNT/C1 binding domain variant will
have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to amino acids
869-1291 of SEQ ID NO: 11; a BoNT/D binding domain variant will
have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to amino acids
865-1291 of SEQ ID NO: 13; a BoNT/E binding domain variant will
have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to amino acids
848-1252 of SEQ ID NO: 15; a BoNT/F binding domain variant will
have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to amino acids
866-1274 of SEQ ID NO: 18; a BoNT/G binding domain variant will
have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to amino acids
866-1297 of SEQ ID NO: 21; a TeNT binding domain variant will have
at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to amino acids
882-1315 of SEQ ID NO: 22; a BaNT binding domain variant will have
at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to amino acids
858-1268 of SEQ ID NO: 23; and a BuNT binding domain variant will
have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to amino acids
848-1251 of SEQ ID NO: 24.
[0156] It is recognized by those of skill in the art that within
each serotype of Clostridial toxin there can be naturally occurring
Clostridial toxin binding domain variants that differ somewhat in
their amino acid sequence, and also in the nucleic acids encoding
these proteins. For example, there are presently five BoNT/A
subtypes, BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, and BoNT/A5, with
specific binding domain subtypes showing about 83-97% amino acid
identity when compared to the BoNT/A binding domain subtype of SEQ
ID NO: 1. As another example, there are presently five BoNT/A
subtypes, BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, and BoNT/A5, with
specific binding domain subtypes showing about 83-97% amino acid
identity when compared to the BoNT/A binding domain subtype of SEQ
ID NO: 1. As used herein, the term "naturally occurring Clostridial
toxin binding domain variant" refers to any Clostridial toxin
binding domain produced by a naturally-occurring process,
including, without limitation, Clostridial toxin binding domain
isoforms produced from alternatively-spliced transcripts,
Clostridial toxin binding domain isoforms produced by spontaneous
mutation and Clostridial toxin binding domain subtypes. A naturally
occurring Clostridial toxin binding domain variant can function in
substantially the same manner as the reference Clostridial toxin
binding domain on which the naturally occurring Clostridial toxin
binding domain variant is based, and can be substituted for the
reference Clostridial toxin binding domain in any aspect of the
present specification.
[0157] A non-limiting examples of a naturally occurring Clostridial
toxin binding domain variant is a Clostridial toxin binding domain
isoform such as, e.g., a BoNT/A binding domain isoform, a BoNT/B
binding domain isoform, a BoNT/C1 binding domain isoform, a BoNT/D
binding domain isoform, a BoNT/E binding domain isoform, a BoNT/F
binding domain isoform, a BoNT/G binding domain isoform, a TeNT
binding domain isoform, a BaNT binding domain isoform, and a BuNT
binding domain isoform. Another non-limiting examples of a
naturally occurring Clostridial toxin binding domain variant is a
Clostridial toxin binding domain subtype such as, e.g., a binding
domain from subtype BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, and
BoNT/A5; a binding domain from subtype BoNT/B1, BoNT/B2, BoNT/B
bivalent and BoNT/B nonproteolytic; a binding domain from subtype
BoNT/C1-1 and BoNT/C1-2; a binding domain from subtype BoNT/E1,
BoNT/E2 and BoNT/E3; and a binding domain from subtype BoNT/F1,
BoNT/F2, and BoNT/F3; and a binding domain from subtype BuNT-1 and
BuNT-2.
[0158] As used herein, the term "non-naturally occurring
Clostridial toxin binding domain variant" refers to any Clostridial
toxin binding domain produced with the aid of human manipulation,
including, without limitation, Clostridial toxin binding domains
produced by genetic engineering using random mutagenesis or
rational design and Clostridial toxin binding domains produced by
chemical synthesis. Non-limiting examples of non-naturally
occurring Clostridial toxin binding domain variants include, e.g.,
conservative Clostridial toxin binding domain variants,
non-conservative Clostridial toxin binding domain variants,
Clostridial toxin binding domain chimeric variants and active
Clostridial toxin binding domain fragments.
[0159] As used herein, the term "conservative Clostridial toxin
binding domain variant" refers to a Clostridial toxin binding
domain that has at least one amino acid substituted by another
amino acid or an amino acid analog that has at least one property
similar to that of the original amino acid from the reference
Clostridial toxin binding domain sequence (Table 1). Examples of
properties include, without limitation, similar size, topography,
charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity, hydrogen-bonding capacity, a
physicochemical property, of the like, or any combination thereof.
A conservative Clostridial toxin binding domain variant can
function in substantially the same manner as the reference
Clostridial toxin binding domain on which the conservative
Clostridial toxin binding domain variant is based, and can be
substituted for the reference Clostridial toxin binding domain in
any aspect of the present specification. Non-limiting examples of a
conservative Clostridial toxin binding domain variant include,
e.g., conservative BoNT/A binding domain variants, conservative
BoNT/B binding domain variants, conservative BoNT/C1 binding domain
variants, conservative BoNT/D binding domain variants, conservative
BoNT/E binding domain variants, conservative BoNT/F binding domain
variants, conservative BoNT/G binding domain variants, conservative
TeNT binding domain variants, conservative BaNT binding domain
variants, and conservative BuNT binding domain variants.
[0160] As used herein, the term "non-conservative Clostridial toxin
binding domain variant" refers to a Clostridial toxin binding
domain in which 1) at least one amino acid is deleted from the
reference Clostridial toxin binding domain on which the
non-conservative Clostridial toxin binding domain variant is based;
2) at least one amino acid added to the reference Clostridial toxin
binding domain on which the non-conservative Clostridial toxin
binding domain is based; or 3) at least one amino acid is
substituted by another amino acid or an amino acid analog that does
not share any property similar to that of the original amino acid
from the reference Clostridial toxin binding domain sequence (Table
1). A non-conservative Clostridial toxin binding domain variant can
function in substantially the same manner as the reference
Clostridial toxin binding domain on which the non-conservative
Clostridial toxin binding domain variant is based, and can be
substituted for the reference Clostridial toxin binding domain in
any aspect of the present specification. Non-limiting examples of a
non-conservative Clostridial toxin binding domain variant include,
e.g., non-conservative BoNT/A binding domain variants,
non-conservative BoNT/B binding domain variants, non-conservative
BoNT/C1 binding domain variants, non-conservative BoNT/D binding
domain variants, non-conservative BoNT/E binding domain variants,
non-conservative BoNT/F binding domain variants, non-conservative
BoNT/G binding domain variants, and non-conservative TeNT binding
domain variants, non-conservative BaNT binding domain variants, and
non-conservative BuNT binding domain variants.
[0161] As used herein, the term "active Clostridial toxin binding
domain fragment" refers to any of a variety of Clostridial toxin
fragments comprising the binding domain can be useful in aspects of
the present specification with the proviso that these active
fragments can facilitate the release of the LC from intracellular
vesicles into the cytoplasm of the target cell and thus participate
in executing the overall cellular mechanism whereby a Clostridial
toxin proteolytically cleaves a substrate. The binding domains from
the heavy chains of Clostridial toxins are approximately 400-440
amino acids in length and comprise a binding domain (Table 1).
Research has shown that the entire length of a binding domain from
a Clostridial toxin heavy chain is not necessary for the
translocating activity of the binding domain. Thus, aspects of this
embodiment include a Clostridial toxin binding domain having a
length of, e.g., at least 350, 375, 400, or 425 amino acids. Other
aspects of this embodiment include a Clostridial toxin binding
domain having a length of, e.g., at most 350, 375, 400, or 425
amino acids.
[0162] Any of a variety of sequence alignment methods can be used
to determine percent identity of naturally-occurring Clostridial
toxin binding domain variants and non-naturally-occurring
Clostridial toxin binding domain variants, including, without
limitation, global methods, local methods, and hybrid methods, such
as, e.g., segment approach methods. Protocols to determine percent
identity are routine procedures within the scope of one skilled in
the art and from the teaching herein.
[0163] Thus, in an embodiment, a modified Clostridial toxin
disclosed in the present specification comprises a Clostridial
toxin binding domain. In an aspect of this embodiment, a
Clostridial toxin binding domain comprises a naturally occurring
Clostridial toxin binding domain variant, such as, e.g., a
Clostridial toxin binding domain isoform or a Clostridial toxin
binding domain subtype. In another aspect of this embodiment, a
Clostridial toxin binding domain comprises a non-naturally
occurring Clostridial toxin binding domain variant, such as, e.g.,
a conservative Clostridial toxin binding domain variant, a
non-conservative Clostridial toxin binding domain variant, an
active Clostridial toxin binding domain fragment, or any
combination thereof.
[0164] In another embodiment, a hydrophobic amino acid at one
particular position in the polypeptide chain of the Clostridial
toxin binding domain can be substituted with another hydrophobic
amino acid. Examples of hydrophobic amino acids include, e.g., C,
F, I, L, M, V and W. In another aspect of this embodiment, an
aliphatic amino acid at one particular position in the polypeptide
chain of the Clostridial toxin binding domain can be substituted
with another aliphatic amino acid. Examples of aliphatic amino
acids include, e.g., A, I, L, P, and V. In yet another aspect of
this embodiment, an aromatic amino acid at one particular position
in the polypeptide chain of the Clostridial toxin binding domain
can be substituted with another aromatic amino acid. Examples of
aromatic amino acids include, e.g., F, H, W and Y. In still another
aspect of this embodiment, a stacking amino acid at one particular
position in the polypeptide chain of the Clostridial toxin binding
domain can be substituted with another stacking amino acid.
Examples of stacking amino acids include, e.g., F, H, W and Y. In a
further aspect of this embodiment, a polar amino acid at one
particular position in the polypeptide chain of the Clostridial
toxin binding domain can be substituted with another polar amino
acid. Examples of polar amino acids include, e.g., D, E, K, N, Q,
and R. In a further aspect of this embodiment, a less polar or
indifferent amino acid at one particular position in the
polypeptide chain of the Clostridial toxin binding domain can be
substituted with another less polar or indifferent amino acid.
Examples of less polar or indifferent amino acids include, e.g., A,
H, G, P, S, T, and Y. In a yet further aspect of this embodiment, a
positive charged amino acid at one particular position in the
polypeptide chain of the Clostridial toxin binding domain can be
substituted with another positive charged amino acid. Examples of
positive charged amino acids include, e.g., K, R, and H. In a still
further aspect of this embodiment, a negative charged amino acid at
one particular position in the polypeptide chain of the Clostridial
toxin binding domain can be substituted with another negative
charged amino acid. Examples of negative charged amino acids
include, e.g., D and E. In another aspect of this embodiment, a
small amino acid at one particular position in the polypeptide
chain of the Clostridial toxin binding domain can be substituted
with another small amino acid. Examples of small amino acids
include, e.g., A, D, G, N, P, S, and T. In yet another aspect of
this embodiment, a C-beta branching amino acid at one particular
position in the polypeptide chain of the Clostridial toxin binding
domain can be substituted with another C-beta branching amino acid.
Examples of C-beta branching amino acids include, e.g., I, T and
V.
[0165] In another embodiment, a Clostridial toxin binding domain
comprises a BoNT/A binding domain. In an aspect of this embodiment,
a BoNT/A binding domain comprises the binding domains of SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In
other aspects of this embodiment, a BoNT/A binding domain comprises
amino acids 874-1296 of SEQ ID NO: 1. In another aspect of this
embodiment, a BoNT/A binding domain comprises a naturally occurring
BoNT/A binding domain variant, such as, e.g., an binding domain
from a BoNT/A isoform or an binding domain from a BoNT/A subtype.
In another aspect of this embodiment, a BoNT/A binding domain
comprises a naturally occurring BoNT/A binding domain variant of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID
NO: 5, such as, e.g., a BoNT/A isoform binding domain or a BoNT/A
subtype binding domain. In another aspect of this embodiment, a
BoNT/A binding domain comprises amino acids 874-1296 of a naturally
occurring BoNT/A binding domain variant of SEQ ID NO: 1, such as,
e.g., a BoNT/A isoform binding domain or a BoNT/A subtype binding
domain. In still another aspect of this embodiment, a BoNT/A
binding domain comprises a non-naturally occurring BoNT/A binding
domain variant, such as, e.g., a conservative BoNT/A binding domain
variant, a non-conservative BoNT/A binding domain variant, an
active BoNT/A binding domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/A binding domain
comprises the binding domain of a non-naturally occurring BoNT/A
binding domain variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., a conservative BoNT/A
binding domain variant, a non-conservative BoNT/A binding domain
variant, an active BoNT/A binding domain fragment, or any
combination thereof. In still another aspect of this embodiment, a
BoNT/A binding domain comprises amino acids 874-1296 of a
non-naturally occurring BoNT/A binding domain variant of SEQ ID NO:
1, such as, e.g., a conservative BoNT/A binding domain variant, a
non-conservative BoNT/A binding domain variant, an active BoNT/A
binding domain fragment, or any combination thereof.
[0166] In other aspects of this embodiment, a BoNT/A binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, or at most 95% to
the binding domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 4, or SEQ ID NO: 5. In yet other aspects of this embodiment,
a BoNT/A binding domain comprises a polypeptide having an amino
acid identity of, e.g., at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, or at least 95% to amino acids 874-1296
of SEQ ID NO: 1; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to amino acids 874-1296 of SEQ ID
NO: 1.
[0167] In other aspects of this embodiment, a BoNT/A binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 non-contiguous amino acid deletions, additions,
and/or substitutions relative to the binding domain of SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In
yet other aspects of this embodiment, a BoNT/A binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
874-1296 of SEQ ID NO: 1; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 874-1296 of
SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A
binding domain comprises a polypeptide having, e.g., at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
binding domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to the binding domain of SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In
further other aspects of this embodiment, a BoNT/A binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 874-1296 of
SEQ ID NO: 1; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 874-1296 of SEQ ID NO: 1.
[0168] In another embodiment, a Clostridial toxin binding domain
comprises a BoNT/B binding domain. In an aspect of this embodiment,
a BoNT/B binding domain comprises the binding domains of SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In
other aspects of this embodiment, a BoNT/B binding domain comprises
amino acids 861-1291 of SEQ ID NO: 6. In another aspect of this
embodiment, a BoNT/B binding domain comprises a naturally occurring
BoNT/B binding domain variant, such as, e.g., an binding domain
from a BoNT/B isoform or an binding domain from a BoNT/B subtype.
In another aspect of this embodiment, a BoNT/B binding domain
comprises a naturally occurring BoNT/B binding domain variant of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID
NO: 10, such as, e.g., a BoNT/B isoform binding domain or a BoNT/B
subtype binding domain. In another aspect of this embodiment, a
BoNT/B binding domain comprises amino acids 861-1291 of a naturally
occurring BoNT/B binding domain variant of SEQ ID NO: 6, such as,
e.g., a BoNT/B isoform binding domain or a BoNT/B subtype binding
domain. In still another aspect of this embodiment, a BoNT/B
binding domain comprises a non-naturally occurring BoNT/B binding
domain variant, such as, e.g., a conservative BoNT/B binding domain
variant, a non-conservative BoNT/B binding domain variant, an
active BoNT/B binding domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/B binding domain
comprises the binding domain of a non-naturally occurring BoNT/B
binding domain variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., a conservative
BoNT/B binding domain variant, a non-conservative BoNT/B binding
domain variant, an active BoNT/B binding domain fragment, or any
combination thereof. In still another aspect of this embodiment, a
BoNT/B binding domain comprises amino acids 861-1291 of a
non-naturally occurring BoNT/B binding domain variant of SEQ ID NO:
6, such as, e.g., a conservative BoNT/B binding domain variant, a
non-conservative BoNT/B binding domain variant, an active BoNT/B
binding domain fragment, or any combination thereof.
[0169] In other aspects of this embodiment, a BoNT/B binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; or at most 70%, at
most 75%, at most 80%, at most 85%, at most 90%, or at most 95% to
the binding domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, or SEQ ID NO: 10. In yet other aspects of this
embodiment, a BoNT/B binding domain comprises a polypeptide having
an amino acid identity of, e.g., at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 861-1291 of SEQ ID NO: 6; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
861-1291 of SEQ ID NO: 6.
[0170] In other aspects of this embodiment, a BoNT/B binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 non-contiguous amino acid deletions, additions,
and/or substitutions relative to the binding domain of SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In
yet other aspects of this embodiment, a BoNT/B binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
861-1291 of SEQ ID NO: 6; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 861-1291 of
SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/B
binding domain comprises a polypeptide having, e.g., at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
binding domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to the binding domain of SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In
further other aspects of this embodiment, a BoNT/B binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 861-1291 of
SEQ ID NO: 6; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 861-1291 of SEQ ID NO: 6.
[0171] In another embodiment, a Clostridial toxin binding domain
comprises a BoNT/C1 binding domain. In an aspect of this
embodiment, a BoNT/C1 binding domain comprises the binding domains
of SEQ ID NO: 11 or SEQ ID NO: 12. In other aspects of this
embodiment, a BoNT/C1 binding domain comprises amino acids 869-1291
of SEQ ID NO: 11. In another aspect of this embodiment, a BoNT/C1
binding domain comprises a naturally occurring BoNT/C1 binding
domain variant, such as, e.g., an binding domain from a BoNT/C1
isoform or an binding domain from a BoNT/C1 subtype. In another
aspect of this embodiment, a BoNT/C1 binding domain comprises a
naturally occurring BoNT/C1 binding domain variant of SEQ ID NO: 11
or SEQ ID NO: 12, such as, e.g., a BoNT/C1 isoform binding domain
or a BoNT/C1 subtype binding domain. In another aspect of this
embodiment, a BoNT/C1 binding domain comprises amino acids 869-1291
of a naturally occurring BoNT/C1 binding domain variant of SEQ ID
NO: 11, such as, e.g., a BoNT/C1 isoform binding domain or a
BoNT/C1 subtype binding domain. In still another aspect of this
embodiment, a BoNT/C1 binding domain comprises a non-naturally
occurring BoNT/C1 binding domain variant, such as, e.g., a
conservative BoNT/C1 binding domain variant, a non-conservative
BoNT/C1 binding domain variant, an active BoNT/C1 binding domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/C1 binding domain comprises the binding
domain of a non-naturally occurring BoNT/C1 binding domain variant
of SEQ ID NO: 11 or SEQ ID NO: 12, such as, e.g., a conservative
BoNT/C1 binding domain variant, a non-conservative BoNT/C1 binding
domain variant, an active BoNT/C1 binding domain fragment, or any
combination thereof. In still another aspect of this embodiment, a
BoNT/C1 binding domain comprises amino acids 869-1291 of a
non-naturally occurring BoNT/C1 binding domain variant of SEQ ID
NO: 11, such as, e.g., a conservative BoNT/C1 binding domain
variant, a non-conservative BoNT/C1 binding domain variant, an
active BoNT/C1 binding domain fragment, or any combination
thereof.
[0172] In other aspects of this embodiment, a BoNT/C1 binding
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to the binding domain of SEQ ID NO: 11
or SEQ ID NO: 12; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to the binding domain of SEQ ID
NO: 11 or SEQ ID NO: 12. In yet other aspects of this embodiment, a
BoNT/C1 binding domain comprises a polypeptide having an amino acid
identity of, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% to amino acids 869-1291 of
SEQ ID NO: 11; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to amino acids 869-1291 of SEQ ID
NO: 11.
[0173] In other aspects of this embodiment, a BoNT/C1 binding
domain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 11 or SEQ ID NO: 12; or at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 11 or SEQ ID NO: 12. In yet other aspects of
this embodiment, a BoNT/C1 binding domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 869-1291 of SEQ ID NO: 11; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 869-1291 of SEQ ID NO: 11. In
still other aspects of this embodiment, a BoNT/C1 binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 11 or SEQ ID NO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 11 or SEQ ID NO: 12. In further other aspects of this
embodiment, a BoNT/C1 binding domain comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 869-1291 of SEQ ID NO: 11; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to amino acids 869-1291 of SEQ ID NO: 11.
[0174] In another embodiment, a Clostridial toxin binding domain
comprises a BoNT/D binding domain. In an aspect of this embodiment,
a BoNT/D binding domain comprises the binding domains of SEQ ID NO:
13 or SEQ ID NO: 14. In other aspects of this embodiment, a BoNT/D
binding domain comprises amino acids 865-1291 of SEQ ID NO: 13. In
another aspect of this embodiment, a BoNT/D binding domain
comprises a naturally occurring BoNT/D binding domain variant, such
as, e.g., an binding domain from a BoNT/D isoform or an binding
domain from a BoNT/D subtype. In another aspect of this embodiment,
a BoNT/D binding domain comprises a naturally occurring BoNT/D
binding domain variant of SEQ ID NO: 13 or SEQ ID NO: 14, such as,
e.g., a BoNT/D isoform binding domain or a BoNT/D subtype binding
domain. In another aspect of this embodiment, a BoNT/D binding
domain comprises amino acids 865-1291 of a naturally occurring
BoNT/D binding domain variant of SEQ ID NO: 13, such as, e.g., a
BoNT/D isoform binding domain or a BoNT/D subtype binding domain.
In still another aspect of this embodiment, a BoNT/D binding domain
comprises a non-naturally occurring BoNT/D binding domain variant,
such as, e.g., a conservative BoNT/D binding domain variant, a
non-conservative BoNT/D binding domain variant, an active BoNT/D
binding domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/D binding domain
comprises the binding domain of a non-naturally occurring BoNT/D
binding domain variant of SEQ ID NO: 13 or SEQ ID NO: 14, such as,
e.g., a conservative BoNT/D binding domain variant, a
non-conservative BoNT/D binding domain variant, an active BoNT/D
binding domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/D binding domain
comprises amino acids 865-1291 of a non-naturally occurring BoNT/D
binding domain variant of SEQ ID NO: 13, such as, e.g., a
conservative BoNT/D binding domain variant, a non-conservative
BoNT/D binding domain variant, an active BoNT/D binding domain
fragment, or any combination thereof.
[0175] In other aspects of this embodiment, a BoNT/D binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 13 or SEQ ID
NO: 14; or at most 70%, at most 75%, at most 80%, at most 85%, at
most 90%, or at most 95% to the binding domain of SEQ ID NO: 13 or
SEQ ID NO: 14. In yet other aspects of this embodiment, a BoNT/D
binding domain comprises a polypeptide having an amino acid
identity of, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% to amino acids 865-1291 of
SEQ ID NO: 13; or at most 70%, at most 75%, at most 80%, at most
85%, at most 90%, or at most 95% to amino acids 865-1291 of SEQ ID
NO: 13.
[0176] In other aspects of this embodiment, a BoNT/D binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 13 or SEQ ID NO: 14. In yet other aspects of
this embodiment, a BoNT/D binding domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 865-1291 of SEQ ID NO: 13; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 865-1291 of SEQ ID NO: 13. In
still other aspects of this embodiment, a BoNT/D binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 13 or SEQ ID NO: 14; or at most 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 13 or SEQ ID NO: 14. In further other aspects of this
embodiment, a BoNT/D binding domain comprises a polypeptide having,
e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 865-1291 of SEQ ID NO: 13; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to amino acids 865-1291 of SEQ ID NO: 13.
[0177] In another embodiment, a Clostridial toxin binding domain
comprises a BoNT/E binding domain. In an aspect of this embodiment,
a BoNT/E binding domain comprises the binding domains of SEQ ID NO:
15, SEQ ID NO: 16, or SEQ ID NO: 17. In other aspects of this
embodiment, a BoNT/E binding domain comprises amino acids 848-1252
of SEQ ID NO: 15. In another aspect of this embodiment, a BoNT/E
binding domain comprises a naturally occurring BoNT/E binding
domain variant, such as, e.g., an binding domain from a BoNT/E
isoform or an binding domain from a BoNT/E subtype. In another
aspect of this embodiment, a BoNT/E binding domain comprises a
naturally occurring BoNT/E binding domain variant of SEQ ID NO: 15,
SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a BoNT/E isoform
binding domain or a BoNT/E subtype binding domain. In another
aspect of this embodiment, a BoNT/E binding domain comprises amino
acids 848-1252 of a naturally occurring BoNT/E binding domain
variant of SEQ ID NO: 15, such as, e.g., a BoNT/E isoform binding
domain or a BoNT/E subtype binding domain. In still another aspect
of this embodiment, a BoNT/E binding domain comprises a
non-naturally occurring BoNT/E binding domain variant, such as,
e.g., a conservative BoNT/E binding domain variant, a
non-conservative BoNT/E binding domain variant, an active BoNT/E
binding domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/E binding domain
comprises the binding domain of a non-naturally occurring BoNT/E
binding domain variant of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID
NO: 17, such as, e.g., a conservative BoNT/E binding domain
variant, a non-conservative BoNT/E binding domain variant, an
active BoNT/E binding domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/E binding domain
comprises amino acids 848-1252 of a non-naturally occurring BoNT/E
binding domain variant of SEQ ID NO: 15, such as, e.g., a
conservative BoNT/E binding domain variant, a non-conservative
BoNT/E binding domain variant, an active BoNT/E binding domain
fragment, or any combination thereof.
[0178] In other aspects of this embodiment, a BoNT/E binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 15, SEQ ID NO:
16, or SEQ ID NO: 17; or at most 70%, at most 75%, at most 80%, at
most 85%, at most 90%, or at most 95% to the binding domain of SEQ
ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In yet other aspects of
this embodiment, a BoNT/E binding domain comprises a polypeptide
having an amino acid identity of, e.g., at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 848-1252 of SEQ ID NO: 15; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
848-1252 of SEQ ID NO: 15.
[0179] In other aspects of this embodiment, a BoNT/E binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to the binding domain of SEQ ID NO: 15, SEQ
ID NO: 16, or SEQ ID NO: 17. In yet other aspects of this
embodiment, a BoNT/E binding domain comprises a polypeptide having,
e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 848-1252 of SEQ ID NO: 15; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 848-1252 of SEQ ID NO: 15. In
still other aspects of this embodiment, a BoNT/E binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or at most 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In
further other aspects of this embodiment, a BoNT/E binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 848-1252 of
SEQ ID NO: 15; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 848-1252 of SEQ ID NO:
15.
[0180] In another embodiment, a Clostridial toxin binding domain
comprises a BoNT/F binding domain. In an aspect of this embodiment,
a BoNT/F binding domain comprises the binding domains of SEQ ID NO:
18, SEQ ID NO: 19, or SEQ ID NO: 20. In other aspects of this
embodiment, a BoNT/F binding domain comprises amino acids 866-1274
of SEQ ID NO: 18. In another aspect of this embodiment, a BoNT/F
binding domain comprises a naturally occurring BoNT/F binding
domain variant, such as, e.g., an binding domain from a BoNT/F
isoform or an binding domain from a BoNT/F subtype. In another
aspect of this embodiment, a BoNT/F binding domain comprises a
naturally occurring BoNT/F binding domain variant of SEQ ID NO: 18,
SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a BoNT/F isoform
binding domain or a BoNT/F subtype binding domain. In another
aspect of this embodiment, a BoNT/F binding domain comprises amino
acids 866-1274 of a naturally occurring BoNT/F binding domain
variant of SEQ ID NO: 18, such as, e.g., a BoNT/F isoform binding
domain or a BoNT/F subtype binding domain. In still another aspect
of this embodiment, a BoNT/F binding domain comprises a
non-naturally occurring BoNT/F binding domain variant, such as,
e.g., a conservative BoNT/F binding domain variant, a
non-conservative BoNT/F binding domain variant, an active BoNT/F
binding domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/F binding domain
comprises the binding domain of a non-naturally occurring BoNT/F
binding domain variant of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID
NO: 20, such as, e.g., a conservative BoNT/F binding domain
variant, a non-conservative BoNT/F binding domain variant, an
active BoNT/F binding domain fragment, or any combination thereof.
In still another aspect of this embodiment, a BoNT/F binding domain
comprises amino acids 866-1274 of a non-naturally occurring BoNT/F
binding domain variant of SEQ ID NO: 18, such as, e.g., a
conservative BoNT/F binding domain variant, a non-conservative
BoNT/F binding domain variant, an active BoNT/F binding domain
fragment, or any combination thereof.
[0181] In other aspects of this embodiment, a BoNT/F binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 18, SEQ ID NO:
19, or SEQ ID NO: 20; or at most 70%, at most 75%, at most 80%, at
most 85%, at most 90%, or at most 95% to the binding domain of SEQ
ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In yet other aspects of
this embodiment, a BoNT/F binding domain comprises a polypeptide
having an amino acid identity of, e.g., at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 866-1274 of SEQ ID NO: 18; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
866-1274 of SEQ ID NO: 18.
[0182] In other aspects of this embodiment, a BoNT/F binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; or at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to the binding domain of SEQ ID NO: 18, SEQ
ID NO: 19, or SEQ ID NO: 20. In yet other aspects of this
embodiment, a BoNT/F binding domain comprises a polypeptide having,
e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 866-1274 of SEQ ID NO: 18; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 866-1274 of SEQ ID NO: 18. In
still other aspects of this embodiment, a BoNT/F binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; or at most 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In
further other aspects of this embodiment, a BoNT/F binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,
additions, and/or substitutions relative to amino acids 866-1274 of
SEQ ID NO: 18; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 866-1274 of SEQ ID NO:
18.
[0183] In another embodiment, a Clostridial toxin binding domain
comprises a BoNT/G binding domain. In an aspect of this embodiment,
a BoNT/G binding domain comprises the binding domains of SEQ ID NO:
21. In other aspects of this embodiment, a BoNT/G binding domain
comprises amino acids 866-1297 of SEQ ID NO: 21. In another aspect
of this embodiment, a BoNT/G binding domain comprises a naturally
occurring BoNT/G binding domain variant, such as, e.g., an binding
domain from a BoNT/G isoform or an binding domain from a BoNT/G
subtype. In another aspect of this embodiment, a BoNT/G binding
domain comprises a naturally occurring BoNT/G binding domain
variant of SEQ ID NO: 21, such as, e.g., a BoNT/G isoform binding
domain or a BoNT/G subtype binding domain. In another aspect of
this embodiment, a BoNT/G binding domain comprises amino acids
866-1297 of a naturally occurring BoNT/G binding domain variant of
SEQ ID NO: 21, such as, e.g., a BoNT/G isoform binding domain or a
BoNT/G subtype binding domain. In still another aspect of this
embodiment, a BoNT/G binding domain comprises a non-naturally
occurring BoNT/G binding domain variant, such as, e.g., a
conservative BoNT/G binding domain variant, a non-conservative
BoNT/G binding domain variant, an active BoNT/G binding domain
fragment, or any combination thereof. In still another aspect of
this embodiment, a BoNT/G binding domain comprises the binding
domain of a non-naturally occurring BoNT/G binding domain variant
of SEQ ID NO: 21, such as, e.g., a conservative BoNT/G binding
domain variant, a non-conservative BoNT/G binding domain variant,
an active BoNT/G binding domain fragment, or any combination
thereof. In still another aspect of this embodiment, a BoNT/G
binding domain comprises amino acids 866-1297 of a non-naturally
occurring BoNT/G binding domain variant of SEQ ID NO: 21, such as,
e.g., a conservative BoNT/G binding domain variant, a
non-conservative BoNT/G binding domain variant, an active BoNT/G
binding domain fragment, or any combination thereof.
[0184] In other aspects of this embodiment, a BoNT/G binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 21; or at most
70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most
95% to the binding domain of SEQ ID NO: 21. In yet other aspects of
this embodiment, a BoNT/G binding domain comprises a polypeptide
having an amino acid identity of, e.g., at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 866-1297 of SEQ ID NO: 21; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
866-1297 of SEQ ID NO: 21.
[0185] In other aspects of this embodiment, a BoNT/G binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 21. In yet other aspects of this embodiment, a BoNT/G
binding domain comprises a polypeptide having, e.g., at most 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid deletions, additions, and/or substitutions relative to
amino acids 866-1297 of SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
866-1297 of SEQ ID NO: 21. In still other aspects of this
embodiment, a BoNT/G binding domain comprises a polypeptide having,
e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to the binding domain of SEQ ID NO: 21; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to the binding domain of SEQ ID NO: 21. In further other
aspects of this embodiment, a BoNT/G binding domain comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 866-1297 of SEQ ID NO:
21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 866-1297 of SEQ ID NO:
21.
[0186] In another embodiment, a Clostridial toxin binding domain
comprises a TeNT binding domain. In an aspect of this embodiment, a
TeNT binding domain comprises the binding domains of SEQ ID NO: 22.
In other aspects of this embodiment, a TeNT binding domain
comprises amino acids 882-1315 of SEQ ID NO: 22. In another aspect
of this embodiment, a TeNT binding domain comprises a naturally
occurring TeNT binding domain variant, such as, e.g., an binding
domain from a TeNT isoform or an binding domain from a TeNT
subtype. In another aspect of this embodiment, a TeNT binding
domain comprises a naturally occurring TeNT binding domain variant
of SEQ ID NO: 22, such as, e.g., a TeNT isoform binding domain or a
TeNT subtype binding domain. In another aspect of this embodiment,
a TeNT binding domain comprises amino acids 882-1315 of a naturally
occurring TeNT binding domain variant of SEQ ID NO: 22, such as,
e.g., a TeNT isoform binding domain or a TeNT subtype binding
domain. In still another aspect of this embodiment, a TeNT binding
domain comprises a non-naturally occurring TeNT binding domain
variant, such as, e.g., a conservative TeNT binding domain variant,
a non-conservative TeNT binding domain variant, an active TeNT
binding domain fragment, or any combination thereof. In still
another aspect of this embodiment, a TeNT binding domain comprises
the binding domain of a non-naturally occurring TeNT binding domain
variant of SEQ ID NO: 22, such as, e.g., a conservative TeNT
binding domain variant, a non-conservative TeNT binding domain
variant, an active TeNT binding domain fragment, or any combination
thereof. In still another aspect of this embodiment, a TeNT binding
domain comprises amino acids 882-1315 of a non-naturally occurring
TeNT binding domain variant of SEQ ID NO: 22, such as, e.g., a
conservative TeNT binding domain variant, a non-conservative TeNT
binding domain variant, an active TeNT binding domain fragment, or
any combination thereof.
[0187] In other aspects of this embodiment, a TeNT binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 22; or at most
70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most
95% to the binding domain of SEQ ID NO: 22. In yet other aspects of
this embodiment, a TeNT binding domain comprises a polypeptide
having an amino acid identity of, e.g., at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 882-1315 of SEQ ID NO: 22; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
882-1315 of SEQ ID NO: 22.
[0188] In other aspects of this embodiment, a TeNT binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 22. In yet other aspects of this embodiment, a TeNT
binding domain comprises a polypeptide having, e.g., at most 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid deletions, additions, and/or substitutions relative to
amino acids 882-1315 of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
882-1315 of SEQ ID NO: 22. In still other aspects of this
embodiment, a TeNT binding domain comprises a polypeptide having,
e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to the binding domain of SEQ ID NO: 22; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to the binding domain of SEQ ID NO: 22. In further other
aspects of this embodiment, a TeNT binding domain comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 882-1315 of SEQ ID NO:
22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 882-1315 of SEQ ID NO:
22.
[0189] In another embodiment, a Clostridial toxin binding domain
comprises a BaNT binding domain. In an aspect of this embodiment, a
BaNT binding domain comprises the binding domains of SEQ ID NO: 23.
In other aspects of this embodiment, a BaNT binding domain
comprises amino acids 858-1268 of SEQ ID NO: 23. In another aspect
of this embodiment, a BaNT binding domain comprises a naturally
occurring BaNT binding domain variant, such as, e.g., an binding
domain from a BaNT isoform or an binding domain from a BaNT
subtype. In another aspect of this embodiment, a BaNT binding
domain comprises a naturally occurring BaNT binding domain variant
of SEQ ID NO: 23, such as, e.g., a BaNT isoform binding domain or a
BaNT subtype binding domain. In another aspect of this embodiment,
a BaNT binding domain comprises amino acids 858-1268 of a naturally
occurring BaNT binding domain variant of SEQ ID NO: 23, such as,
e.g., a BaNT isoform binding domain or a BaNT subtype binding
domain. In still another aspect of this embodiment, a BaNT binding
domain comprises a non-naturally occurring BaNT binding domain
variant, such as, e.g., a conservative BaNT binding domain variant,
a non-conservative BaNT binding domain variant, an active BaNT
binding domain fragment, or any combination thereof. In still
another aspect of this embodiment, a BaNT binding domain comprises
the binding domain of a non-naturally occurring BaNT binding domain
variant of SEQ ID NO: 23, such as, e.g., a conservative BaNT
binding domain variant, a non-conservative BaNT binding domain
variant, an active BaNT binding domain fragment, or any combination
thereof. In still another aspect of this embodiment, a BaNT binding
domain comprises amino acids 858-1268 of a non-naturally occurring
BaNT binding domain variant of SEQ ID NO: 23, such as, e.g., a
conservative BaNT binding domain variant, a non-conservative BaNT
binding domain variant, an active BaNT binding domain fragment, or
any combination thereof.
[0190] In other aspects of this embodiment, a BaNT binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 23; or at most
70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most
95% to the binding domain of SEQ ID NO: 23. In yet other aspects of
this embodiment, a BaNT binding domain comprises a polypeptide
having an amino acid identity of, e.g., at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95% to amino
acids 858-1268 of SEQ ID NO: 23; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
858-1268 of SEQ ID NO: 23.
[0191] In other aspects of this embodiment, a BaNT binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 non-contiguous amino acid deletions,
additions, and/or substitutions relative to the binding domain of
SEQ ID NO: 23. In yet other aspects of this embodiment, a BaNT
binding domain comprises a polypeptide having, e.g., at most 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid deletions, additions, and/or substitutions relative to
amino acids 858-1268 of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to amino acids
858-1268 of SEQ ID NO: 23. In still other aspects of this
embodiment, a BaNT binding domain comprises a polypeptide having,
e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to the binding domain of SEQ ID NO: 23; or
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100
contiguous amino acid deletions, additions, and/or substitutions
relative to the binding domain of SEQ ID NO: 23. In further other
aspects of this embodiment, a BaNT binding domain comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 858-1268 of SEQ ID NO:
23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or
100 contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 858-1268 of SEQ ID NO:
23.
[0192] In another embodiment, a Clostridial toxin binding domain
comprises a BuNT binding domain. In an aspect of this embodiment, a
BuNT binding domain comprises the binding domains of SEQ ID NO: 24
or SEQ ID NO: 25. In other aspects of this embodiment, a BuNT
binding domain comprises amino acids 848-1251 of SEQ ID NO: 24. In
another aspect of this embodiment, a BuNT binding domain comprises
a naturally occurring BuNT binding domain variant, such as, e.g.,
an binding domain from a BuNT isoform or an binding domain from a
BuNT subtype. In another aspect of this embodiment, a BuNT binding
domain comprises a naturally occurring BuNT binding domain variant
of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g., a BuNT isoform
binding domain or a BuNT subtype binding domain. In another aspect
of this embodiment, a BuNT binding domain comprises amino acids
848-1251 of a naturally occurring BuNT binding domain variant of
SEQ ID NO: 24, such as, e.g., a BuNT isoform binding domain or a
BuNT subtype binding domain. In still another aspect of this
embodiment, a BuNT binding domain comprises a non-naturally
occurring BuNT binding domain variant, such as, e.g., a
conservative BuNT binding domain variant, a non-conservative BuNT
binding domain variant, an active BuNT binding domain fragment, or
any combination thereof. In still another aspect of this
embodiment, a BuNT binding domain comprises the binding domain of a
non-naturally occurring BuNT binding domain variant of SEQ ID NO:
24 or SEQ ID NO: 25, such as, e.g., a conservative BuNT binding
domain variant, a non-conservative BuNT binding domain variant, an
active BuNT binding domain fragment, or any combination thereof. In
still another aspect of this embodiment, a BuNT binding domain
comprises amino acids 848-1251 of a non-naturally occurring BuNT
binding domain variant of SEQ ID NO: 24, such as, e.g., a
conservative BuNT binding domain variant, a non-conservative BuNT
binding domain variant, an active BuNT binding domain fragment, or
any combination thereof.
[0193] In other aspects of this embodiment, a BuNT binding domain
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to the binding domain of SEQ ID NO: 24 or SEQ ID
NO: 25; or at most 70%, at most 75%, at most 80%, at most 85%, at
most 90%, or at most 95% to the binding domain of SEQ ID NO: 24 or
SEQ ID NO: 25. In yet other aspects of this embodiment, a BuNT
binding domain comprises a polypeptide having an amino acid
identity of, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% to amino acids 848-1251 of
SEQ ID NO: 24 or SEQ ID NO: 25; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
848-1251 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0194] In other aspects of this embodiment, a BuNT binding domain
comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
deletions, additions, and/or substitutions relative to the binding
domain of SEQ ID NO: 24 OR SEQ ID NO: 25. In yet other aspects of
this embodiment, a BuNT binding domain comprises a polypeptide
having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 848-1251 of SEQ ID NO: 24 or
SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, or 100 non-contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 848-1251 of SEQ ID NO:
24 or SEQ ID NO: 25. In still other aspects of this embodiment, a
BuNT binding domain comprises a polypeptide having, e.g., at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous
amino acid deletions, additions, and/or substitutions relative to
the binding domain of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino
acid deletions, additions, and/or substitutions relative to the
binding domain of SEQ ID NO: 24 or SEQ ID NO: 25. In further other
aspects of this embodiment, a BuNT binding domain comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 848-1251 of SEQ ID NO:
24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, or 100 contiguous amino acid deletions, additions,
and/or substitutions relative to amino acids 848-1251 of SEQ ID NO:
24 or SEQ ID NO: 25.
[0195] Aspects of the present specification provide, in part, a
non-Clostridial toxin binding domain. As used herein, the term
"non-Clostridial toxin binding domain" refers to any polypeptide
that can execute the binding step of the intoxication process that
initiates the overall internalization mechanism whereby the
modified Clostridial toxin disclosed in the present specification
intoxicates a target cell. Examples of binding domains are
described in, e.g., Keith A. Foster et al., Clostridial Toxin
Derivatives Able To Modify Peripheral Sensory Afferent Functions,
U.S. Pat. No. 5,989,545; Clifford C. Shone et al., Recombinant
Toxin Fragments, U.S. Pat. No. 6,461,617; Conrad P. Quinn et al.,
Methods and Compounds for the Treatment of Mucus Hypersecretion,
U.S. Pat. No. 6,632,440; Lance E. Steward et al., Methods And
Compositions For The Treatment Of Pancreatitis, U.S. Pat. No.
6,843,998; J. Oliver Dolly et al., Activatable Recombinant
Neurotoxins, U.S. Pat. No. 7,132,259; Stephan Donovan, Clostridial
Toxin Derivatives and Methods For Treating Pain, U.S. Patent
Publication 2002/0037833; Keith A. Foster et al., Inhibition of
Secretion from Non-neural Cells, U.S. Patent Publication
2003/0180289; Lance E. Steward et al., Multivalent Clostridial
Toxin Derivatives and Methods of Their Use, U.S. Patent Publication
2006/0211619; Keith A. Foster et al., Non-Cytotoxic Protein
Conjugates, U.S. Patent Publication 2008/0187960; Steward, L. E. et
al., Modified Clostridial Toxins with Enhanced Translocation
Capabilities and Altered Targeting Activity For Non-Clostridial
Toxin Target Cells, U.S. patent application Ser. No. 11/776,075;
Keith A. Foster et al., Re-targeted Toxin Conjugates, U.S. patent
application Ser. No. 11/792,210; each of which is incorporated by
reference in its entirety.
[0196] A non-Clostridial toxin binding domain includes, without
limitation, naturally occurring non-Clostridial toxin binding
domain variants, such as, e.g., non-Clostridial toxin binding
domain isoforms and non-Clostridial toxin binding domain subtypes;
and non-naturally occurring non-Clostridial toxin binding domain
variants, such as, e.g., conservative non-Clostridial toxin binding
domain variants, non-conservative non-Clostridial toxin binding
domain variants, non-Clostridial toxin binding domain chimeras,
active non-Clostridial toxin binding domain fragments thereof, or
any combination thereof.
[0197] As used herein, the term "non-Clostridial toxin binding
domain variant," whether naturally-occurring or
non-naturally-occurring, refers to a non-Clostridial toxin binding
domain that has at least one amino acid change from the
corresponding region of a reference sequence and can be described
in percent identity to the corresponding region of that reference
sequence. Unless expressly indicated, non-Clostridial toxin binding
domain variants useful to practice the disclosed embodiments are
variants that execute the binding step of the intoxication
process.
[0198] It is recognized by those of skill in the art that within
each non-Clostridial toxin binding domain there can be naturally
occurring variants that differ somewhat in their amino acid
sequence, and also in the nucleic acids encoding these proteins. As
used herein, the term "naturally occurring non-Clostridial toxin
binding domain variant" refers to any non-Clostridial toxin binding
domain produced by a naturally-occurring process, including,
without limitation, non-Clostridial toxin binding domain isoforms
produced from alternatively-spliced transcripts and non-Clostridial
toxin binding domain isoforms produced by spontaneous mutation. A
naturally occurring non-Clostridial toxin binding domain variant
can function in substantially the same manner as the reference
non-Clostridial toxin binding domain on which the naturally
occurring non-Clostridial toxin binding domain variant is based,
and can be substituted for the reference non-Clostridial toxin
binding domain in any aspect of the present specification. A
non-limiting examples of a naturally occurring non-Clostridial
toxin binding domain variant is a non-Clostridial toxin binding
domain isoform. Non-limiting examples of a non-Clostridial toxin
binding domain isoform include, e.g., opioid binding domain
isoforms, tachykinin binding domain isoforms, melanocortin binding
domain isoforms, galanin binding domain isoforms, granin binding
domain isoforms, Neuropeptide Y related peptide binding domain
isoforms, neurohormone binding domain isoforms, neuroregulatory
cytokine binding domain isoforms, kinin peptide binding domain
isoforms, growth factor binding domain isoforms, and glucagon like
hormone binding domain isoforms.
[0199] As used herein, the term "non-naturally occurring
non-Clostridial toxin binding domain variant" refers to any
non-Clostridial toxin binding domain produced with the aid of human
manipulation, including, without limitation, non-Clostridial toxin
binding domains produced by genetic engineering using random
mutagenesis or rational design and non-Clostridial toxin binding
domains produced by chemical synthesis. Non-limiting examples of
non-naturally occurring non-Clostridial toxin binding domain
variants include, e.g., conservative non-Clostridial toxin binding
domain variants, non-conservative non-Clostridial toxin binding
domain variants, non-Clostridial toxin binding domain chimeric
variants and active non-Clostridial toxin binding domain
fragments.
[0200] As used herein, the term "conservative non-Clostridial toxin
binding domain variant" refers to a non-Clostridial toxin binding
domain that has at least one amino acid substituted by another
amino acid or an amino acid analog that has at least one property
similar to that of the original amino acid from a reference
non-Clostridial toxin binding domain sequence. Examples of
properties include, without limitation, similar size, topography,
charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity, hydrogen-bonding capacity, a
physicochemical property, of the like, or any combination thereof.
A conservative non-Clostridial toxin binding domain variant can
function in substantially the same manner as the reference
non-Clostridial toxin binding domain on which the conservative
non-Clostridial toxin binding domain variant is based, and can be
substituted for the reference non-Clostridial toxin binding domain
in any aspect of the present specification. Non-limiting examples
of a conservative non-Clostridial toxin binding domain variant
include, e.g., conservative opioid binding domain variants,
conservative tachykinin binding domain variants, conservative
melanocortin binding domain variants, conservative galanin binding
domain variants, conservative granin binding domain variants,
conservative Neuropeptide Y related peptide binding domain
variants, conservative neurohormone binding domain variants,
conservative neuroregulatory cytokine binding domain variants,
conservative kinin peptide binding domain variants, conservative
growth factor binding domain variants, and conservative glucagon
like hormone binding domain variants.
[0201] As used herein, the term "non-conservative non-Clostridial
toxin binding domain variant" refers to a non-Clostridial toxin
binding domain in which 1) at least one amino acid is deleted from
the reference non-Clostridial toxin binding domain on which the
non-conservative non-Clostridial toxin binding domain variant is
based; 2) at least one amino acid added to the reference
non-Clostridial toxin binding domain on which the non-conservative
non-Clostridial toxin binding domain is based; or 3) at least one
amino acid is substituted by another amino acid or an amino acid
analog that does not share any property similar to that of the
original amino acid from a reference non-Clostridial toxin binding
domain sequence. A non-conservative non-Clostridial toxin binding
domain variant can function in substantially the same manner as the
reference non-Clostridial toxin binding domain on which the
non-conservative non-Clostridial toxin binding domain variant is
based, and can be substituted for the reference non-Clostridial
toxin binding domain in any aspect of the present specification.
Non-limiting examples of a non-conservative non-Clostridial toxin
binding domain variant include, e.g., non-conservative opioid
binding domain variants, non-conservative tachykinin binding domain
variants, non-conservative melanocortin binding domain variants,
non-conservative galanin binding domain variants, non-conservative
granin binding domain variants, non-conservative Neuropeptide Y
related peptide binding domain variants, non-conservative
neurohormone binding domain variants, non-conservative
neuroregulatory cytokine binding domain variants, non-conservative
kinin peptide binding domain variants, non-conservative growth
factor binding domain variants, and non-conservative glucagon like
hormone binding domain variants.
[0202] As used herein, the term "active non-Clostridial toxin
binding domain fragment" refers to any of a variety of Clostridial
toxin fragments comprising the binding domain can be useful in
aspects of the present specification with the proviso that these
biding domain fragments can preferentially interact with the
cognate receptor, and thus participate in executing the overall
cellular mechanism whereby a Clostridial toxin proteolytically
cleaves a substrate.
[0203] Any of a variety of sequence alignment methods can be used
to determine percent identity of naturally-occurring Clostridial
toxin binding domain variants and non-naturally-occurring
Clostridial toxin binding domain variants, including, without
limitation, global methods, local methods and hybrid methods, such
as, e.g., segment approach methods. Protocols to determine percent
identity are routine procedures within the scope of one skilled in
the art and from the teaching herein.
[0204] Thus, in an embodiment, a modified Clostridial toxin
disclosed in the present specification comprises a non-Clostridial
toxin binding domain. In an aspect of this embodiment, a
non-Clostridial toxin binding domain comprises a naturally
occurring non-Clostridial toxin binding domain variant, such as,
e.g., a non-Clostridial toxin binding domain isoform. In another
aspect of this embodiment, a non-Clostridial toxin binding domain
comprises a non-naturally occurring non-Clostridial toxin binding
domain variant, such as, e.g., a conservative non-Clostridial toxin
binding domain variant, a non-conservative non-Clostridial toxin
binding domain variant, an active non-Clostridial toxin binding
domain fragment, or any combination thereof.
[0205] In another embodiment, a hydrophobic amino acid at one
particular position in the polypeptide chain of the non-Clostridial
toxin binding domain can be substituted with another hydrophobic
amino acid. Examples of hydrophobic amino acids include, e.g., C,
F, I, L, M, V and W. In another aspect of this embodiment, an
aliphatic amino acid at one particular position in the polypeptide
chain of the non-Clostridial toxin binding domain can be
substituted with another aliphatic amino acid. Examples of
aliphatic amino acids include, e.g., A, I, L, P, and V. In yet
another aspect of this embodiment, an aromatic amino acid at one
particular position in the polypeptide chain of the non-Clostridial
toxin binding domain can be substituted with another aromatic amino
acid. Examples of aromatic amino acids include, e.g., F, H, W and
Y. In still another aspect of this embodiment, a stacking amino
acid at one particular position in the polypeptide chain of the
non-Clostridial toxin binding domain can be substituted with
another stacking amino acid. Examples of stacking amino acids
include, e.g., F, H, W and Y. In a further aspect of this
embodiment, a polar amino acid at one particular position in the
polypeptide chain of the non-Clostridial toxin binding domain can
be substituted with another polar amino acid. Examples of polar
amino acids include, e.g., D, E, K, N, Q, and R. In a further
aspect of this embodiment, a less polar or indifferent amino acid
at one particular position in the polypeptide chain of the
non-Clostridial toxin binding domain can be substituted with
another less polar or indifferent amino acid. Examples of less
polar or indifferent amino acids include, e.g., A, H, G, P, S, T,
and Y. In a yet further aspect of this embodiment, a positive
charged amino acid at one particular position in the polypeptide
chain of the non-Clostridial toxin binding domain can be
substituted with another positive charged amino acid. Examples of
positive charged amino acids include, e.g., K, R, and H. In a still
further aspect of this embodiment, a negative charged amino acid at
one particular position in the polypeptide chain of the
non-Clostridial toxin binding domain can be substituted with
another negative charged amino acid. Examples of negative charged
amino acids include, e.g., D and E. In another aspect of this
embodiment, a small amino acid at one particular position in the
polypeptide chain of the non-Clostridial toxin binding domain can
be substituted with another small amino acid. Examples of small
amino acids include, e.g., A, D, G, N, P, S, and T. In yet another
aspect of this embodiment, a C-beta branching amino acid at one
particular position in the polypeptide chain of the non-Clostridial
toxin binding domain can be substituted with another C-beta
branching amino acid. Examples of C-beta branching amino acids
include, e.g., I, T and V.
[0206] In another embodiment, a non-Clostridial toxin binding
domain comprises an opioid binding domain, such as, e.g., an
enkephalin, an endomorphin, an endorphin, a dynorphin, a nociceptin
or a hemorphin. In yet another aspect of this embodiment, a
non-Clostridial toxin binding domain comprises a tachykinin binding
domain, such as, e.g., a Substance P, a neuropeptide K (NPK), a
neuropeptide gamma (NP gamma), a neurokinin A (NKA; Substance K,
neurokinin alpha, neuromedin L), a neurokinin B (NKB), a hemokinin
or a endokinin. In still another aspect of this embodiment, a
non-Clostridial toxin comprises a melanocortin binding domain, such
as, e.g., a melanocyte stimulating hormone, adrenocorticotropin, or
a lipotropin. In still another aspect of this embodiment, a
non-Clostridial toxin binding domain comprises a galanin binding
domain, such as, e.g., a galanin or a galanin message-associated
peptide. In a further aspect of this embodiment, a non-Clostridial
toxin binding domain comprises a granin binding domain, such as,
e.g., a Chromogranin A, a Chromogranin B, or a Chromogranin C. In
another aspect of this embodiment, a non-Clostridial toxin binding
domain comprises a Neuropeptide Y related peptide binding domain,
such as, e.g., a Neuropeptide Y, a Peptide YY, Pancreatic peptide
or a Pancreatic icosapeptide. In yet another aspect of this
embodiment, a non-Clostridial toxin binding domain comprises a
neurohormone binding domain, such as, e.g., a
corticotropin-releasing hormone, a parathyroid hormone, a
thyrotropin-releasing hormone, or a somatostatin. In still another
aspect of this embodiment, a non-Clostridial toxin binding domain
comprises a neuroregulatory cytokine binding domain, such as, e.g.,
a ciliary neurotrophic factor, a glycophorin-A, a leukemia
inhibitory factor, a cholinergic differentiation factor, an
interleukin, an oncostatin M, a cardiotrophin-1, a
cardiotrophin-like cytokine, or a neuroleukin. In a further aspect
of this embodiment, a non-Clostridial toxin binding domain
comprises a kinin peptide binding domain, such as, e.g., a
bradykinin, a kallidin, a desArg9 bradykinin, or a desArg10
bradykinin. In another aspect of this embodiment, a non-Clostridial
toxin binding domain comprises a growth factor binding domain, such
as, e.g., a fibroblast growth factor binding domain, a nerve growth
factor binding domain, an insulin growth factor binding domain, an
epidermal growth factor binding domain, a vascular endothelial
growth factor binding domain, a brain derived neurotrophic factor
binding domain, a growth derived neurotrophic factor binding
domain, a neurotrophin binding domain, such as, e.g., a
neurotrophin-3, a neurotrophin-4/5, a head activator peptide
binding domain, a neurturin binding domain, a persephin binding
domain, an artemin binding domain, a transformation growth factor
.beta. binding domain, such as, e.g., a TGF.beta.1, a TGF.beta.2, a
TGF.beta.3 or a TGF.beta.4, a bone morphogenic protein binding
domain, such as, e.g., a BMP2, a BMP3, a BMP4, a BMP5, a BMP6, a
BMP7, a BMP8 or a BMP10, a growth differentiation factor binding
domain, such as, e.g., a GDF1, a GDF2, a GDF3, a GDF5, a GDF6, a
GDF7, a GDF8, a GDF10, a GDF11 or a GDF15, or an activin binding
domain, such as, e.g., an activin A, an activin B, an activin C, an
activin E or an inhibin A. In another aspect of this embodiment, a
non-Clostridial toxin binding domain comprises a glucagon like
hormone binding domain, such as, e.g., a secretin, a glucagon-like
peptide, like a GLP-1 and a GLP-2, a pituitary adenylate cyclase
activating peptide binding domain, a growth hormone-releasing
hormone binding domain, vasoactive intestinal peptide binding
domain like a VIP1 or a VIP2, a gastric inhibitory polypeptide
binding domain, a calcitonin-related peptide, visceral gut peptide
binding domain like a gastrin, a gastrin-releasing peptide or a
cholecystokinin, or a PAR peptide binding domain like a PAR1
peptide, a PAR2 peptide, a PAR3 peptide or a PAR4 peptide.
[0207] In another embodiment, an opioid peptide comprises an
enkephalin peptide. In aspects of this embodiment, an enkephalin
peptide comprises a Leu-enkephalin, a Met-enkephalin, a
Met-enkephalin MRGL or a Met-enkephalin MRF. In other aspects of
this embodiment, an enkephalin peptide comprises SEQ ID NO: 26, SEQ
ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.
[0208] In other aspects of this embodiment, an enkephalin comprises
a polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO:
29; or at most 70%, at most 75%, at most 80%, at most 85%, at most
90%, or at most 95% to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28
or SEQ ID NO: 29. In yet other aspects of this embodiment, an
enkephalin comprises a polypeptide having, e.g., at least 1, 2, or
3 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
28 or SEQ ID NO: 29; or at most 1, 2, or 3 non-contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. In still
other aspects of this embodiment, an enkephalin comprises a
polypeptide having, e.g., at least 1, 2, or 3 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29; or at most 1, 2,
or 3 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
28 or SEQ ID NO: 29.
[0209] In another embodiment, an opioid peptide comprises a bovine
adrenomedullary-22 (BAM22) peptide. In aspects of this embodiment,
a BAM22 peptide comprises a BAM22 peptide (1-12), a BAM22 peptide
(6-22), a BAM22 peptide (8-22) or a BAM22 peptide (1-22). In other
aspects of this embodiment, a BAM22 peptide comprises amino acids
1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ
ID NO: 30; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 31; amino acids 1-12, amino acids
6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 32; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 33; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-22 of SEQ ID NO: 34 or amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 35.
[0210] In other aspects of this embodiment, a BAM22 peptide
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-22 of SEQ ID NO: 30; amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 31;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO: 32; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 33; amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 34 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 35; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to amino acids
1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ
ID NO: 30; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 31; amino acids 1-12, amino acids
6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 32; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 33; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-22 of SEQ ID NO: 34 or amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 35.
[0211] In yet other aspects of this embodiment, a BAM22 peptide
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, or 5
non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 30; amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 31; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 32; amino acids 1-12, amino acids
6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 33; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 34 or amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-22 of SEQ ID NO: 35; or at most 1, 2, 3, 4,
or 5 non-contiguous amino acid deletions, additions, and/or
substitutions relative to amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 30; amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 31; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 32; amino acids 1-12, amino acids
6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 33; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 34 or amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-22 of SEQ ID NO: 35.
[0212] In still other aspects of this embodiment, a BAM22 peptide
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, or 5
contiguous amino acid deletions, additions, and/or substitutions
relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 30; amino acids 1-12, amino acids
6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 31; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 32; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-22 of SEQ ID NO: 33; amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 34
or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 35; or at most 1, 2, 3, 4, or 5 contiguous
amino acid deletions, additions, and/or substitutions relative to
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO: 30; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 71; amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 32; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 33; amino acids 1-12, amino acids
6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 34 or
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO: 35.
[0213] In another embodiment, an opioid peptide comprises an
endomorphin peptide. In aspects of this embodiment, an endomorphin
peptide comprises an endomorphin-1 or an endomorphin-2. In other
aspects of this embodiment, an endomorphin peptide comprises SEQ ID
NO: 36 or SEQ ID NO: 37.
[0214] In other aspects of this embodiment, an endomorphin
comprises a polypeptide having an amino acid identity of, e.g., at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% to SEQ ID NO: 36 or SEQ ID NO: 37; or at most 70%,
at most 75%, at most 80%, at most 85%, at most 90%, or at most 95%
to SEQ ID NO: 36 or SEQ ID NO: 37. In yet other aspects of this
embodiment, an endomorphin comprises a polypeptide having, e.g., at
least 1, 2, or 3 non-contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 36 or SEQ ID NO: 37; or
at most 1, 2, or 3 non-contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 36 or SEQ ID NO: 37. In
still other aspects of this embodiment, an endomorphin comprises a
polypeptide having, e.g., at least 1, 2, or 3 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
36 or SEQ ID NO: 37; or at most 1, 2, or 3 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
36 or SEQ ID NO: 37.
[0215] In another embodiment, an opioid peptide comprises an
endorphin peptide. In aspects of this embodiment, an endorphin
peptide comprises an endorphin-.alpha., a neoendorphin-.alpha., an
endorphin-.beta., a neoendorphin-.beta. or an endorphin-.gamma.. In
other aspects of this embodiment, an endorphin peptide comprises
SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
NO: 42, or SEQ ID NO: 43.
[0216] In other aspects of this embodiment, an endorphin comprises
a polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,
SEQ ID NO: 42, or SEQ ID NO: 43; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO:
38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or
SEQ ID NO: 43. In yet other aspects of this embodiment, an
endorphin comprises a polypeptide having, e.g., at least 1, 2, 3,
4, or 5 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:
40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43; or at most 1,
2, 3, 4, or 5 non-contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 38, SEQ ID NO: 39, SEQ
ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. In still
other aspects of this embodiment, an endorphin comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, or 5 contiguous
amino acid deletions, additions, and/or substitutions relative to
SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
NO: 42, or SEQ ID NO: 43; or at most 1, 2, 3, 4, or 5 contiguous
amino acid deletions, additions, and/or substitutions relative to
SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
NO: 42, or SEQ ID NO: 43.
[0217] In another embodiment, an opioid peptide comprises a
dynorphin peptide. In aspects of this embodiment, a dynorphin
peptide comprises a dynorphin A, a dynorphin B (leumorphin) or a
rimorphin. In other aspects of this embodiment, a dynorphin peptide
comprises SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:
47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ
ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:
56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ
ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:
65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ
ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID
NO: 74.
[0218] In other aspects of this embodiment, a dynorphin comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,
SEQ ID NO: 53, or SEQ ID NO: 69; or at most 70%, at most 75%, at
most 80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO:
44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 53, or
SEQ ID NO: 69. In yet other aspects of this embodiment, a dynorphin
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:
46, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO: 69; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 44, SEQ ID
NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO:
69. In still other aspects of this embodiment, a dynorphin
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:
46, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO: 69; or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 44, SEQ ID
NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO:
69.
[0219] In another embodiment, an opioid peptide comprises a
nociceptin peptide. In aspects of this embodiment, a nociceptin
peptide comprises a nociceptin RK, a nociceptin, a neuropeptide 1,
a neuropeptide 2, or a neuropeptide 3. In other aspects of this
embodiment, a nociceptin peptide comprises SEQ ID NO: 75, SEQ ID
NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80,
SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84.
[0220] In other aspects of this embodiment, a nociceptin comprises
a polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,
SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID
NO: 83, or SEQ ID NO: 84; or at most 70%, at most 75%, at most 80%,
at most 85%, at most 90%, or at most 95% to SEQ ID NO: 75, SEQ ID
NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80,
SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84. In
yet other aspects of this embodiment, a nociceptin comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:
77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ
ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84; or at most 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 75, SEQ ID
NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80,
SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84. In
still other aspects of this embodiment, a nociceptin comprises a
polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
contiguous amino acid deletions, additions, and/or substitutions
relative to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:
78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ
ID NO: 83, or SEQ ID NO: 84; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:
77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ
ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84.
[0221] In another embodiment, an opioid peptide comprises a
hemorphin peptide. In aspects of this embodiment, a hemorphin
peptide comprises a LVVH7, a VVH7, a VH7, a H7, a LVVH6, a LVVH5, a
VVH5, a LVVH4, and a LVVH3. In other aspects of this embodiment, a
hemorphin peptide comprises SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID
NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91,
SEQ ID NO: 92, or SEQ ID NO: 93.
[0222] In other aspects of this embodiment, a hemorphin comprises a
polypeptide having an amino acid identity of, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88,
SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ
ID NO: 93; or at most 70%, at most 75%, at most 80%, at most 85%,
at most 90%, or at most 95% to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID
NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91,
SEQ ID NO: 92, or SEQ ID NO: 93. In yet other aspects of this
embodiment, a nociceptin comprises a polypeptide having, e.g., at
least 1, 2, or 3 non-contiguous amino acid deletions, additions,
and/or substitutions relative to SEQ ID NO: 85, SEQ ID NO: 86, SEQ
ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO:
91, SEQ ID NO: 92, or SEQ ID NO: 93; or at most 1, 2, or 3
non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO:
87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ
ID NO: 92, or SEQ ID NO: 93. In still other aspects of this
embodiment, a nociceptin comprises a polypeptide having, e.g., at
least 1, 2, or 3 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO:
87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ
ID NO: 92, or SEQ ID NO: 93; or at most 1, 2, or 3 contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93.
[0223] In yet another embodiment, a non-Clostridial toxin binding
domain comprises a galanin peptide binding domain. In aspects of
this embodiment, a galanin peptide binding domain comprises a
galanin or a galanin message-associated peptide (GMAP). In other
aspects of this embodiment, a galanin peptide binding domain
comprises SEQ ID NO: 94 or SEQ ID NO: 95.
[0224] In other aspects of this embodiment, a galanin binding
domain comprises a polypeptide having an amino acid identity of,
e.g., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% to SEQ ID NO: 94 or SEQ ID NO: 95; or at
most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at
most 95% to SEQ ID NO: 94 or SEQ ID NO: 95. In yet other aspects of
this embodiment, a galanin binding domain comprises a polypeptide
having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 94 or SEQ ID NO: 95; or at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
94 or SEQ ID NO: 95. In still other aspects of this embodiment, a
galanin binding domain comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
94 or SEQ ID NO: 95; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
contiguous amino acid deletions, additions, and/or substitutions
relative to SEQ ID NO: 94 or SEQ ID NO: 95.
[0225] In still another embodiment, a non-Clostridial toxin binding
domain comprises a tachykinin peptide binding domain. In aspects of
this embodiment, a tachykinin peptide binding domain comprises a
Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NP
gamma), a neurokinin A (NKA; Substance K, neurokinin alpha,
neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin. In
other aspects of this embodiment, a tachykinin peptide binding
domain comprises SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ
ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID
NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID
NO: 107.
[0226] In other aspects of this embodiment, a tachykinin peptide
binding domain comprises a polypeptide having an amino acid
identity of, e.g., at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% to SEQ ID NO: 96, SEQ ID
NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO:
101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:
105, SEQ ID NO: 106, or SEQ ID NO: 107; or at most 70%, at most
75%, at most 80%, at most 85%, at most 90%, or at most 95% to SEQ
ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:
100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107. In yet
other aspects of this embodiment, a tachykinin peptide binding
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
or 5 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:
98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or
SEQ ID NO: 107; or at most 1, 2, 3, 4, or 5 non-contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:
100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107. In still
other aspects of this embodiment, a tachykinin peptide binding
domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4,
or 5 contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:
98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or
SEQ ID NO: 107; or at most 1, 2, 3, 4, or 5 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100,
SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ
ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107.
[0227] In still another embodiment, a non-Clostridial toxin binding
domain comprises a Neuropeptide Y related peptide binding domain.
In aspects of this embodiment, a Neuropeptide Y related peptide
binding domain comprises a Neuropeptide Y (NPY), a Peptide YY
(PYY), Pancreatic peptide (PP) or a Pancreatic icosapeptide (PIP).
In other aspects of this embodiment, a Neuropeptide Y related
peptide binding domain comprises SEQ ID NO: 108, SEQ ID NO: 109,
SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112.
[0228] In other aspects of this embodiment, a Neuropeptide Y
related peptide binding domain comprises a polypeptide having an
amino acid identity of, e.g., at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 108,
SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112;
or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,
or at most 95% to SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,
SEQ ID NO: 111, or SEQ ID NO: 112. In yet other aspects of this
embodiment, a Neuropeptide Y related peptide binding domain
comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 non-contiguous amino acid deletions, additions, and/or
substitutions relative to SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112; or at most 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 non-contiguous amino acid deletions,
additions, and/or substitutions relative to SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112. In
still other aspects of this embodiment, a Neuropeptide Y related
peptide binding domain comprises a polypeptide having, e.g., at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid
deletions, additions, and/or substitutions relative to SEQ ID NO:
108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO:
112; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino
acid deletions, additions, and/or substitutions relative to SEQ ID
NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID
NO: 112.
[0229] It is envisioned that a Clostridial toxin chimeric disclosed
in the present specification can comprise a non-Clostridial binding
domain in any and all locations with the proviso that Clostridial
toxin chimeric can perform the intoxication process. Non-limiting
examples include, locating a non-Clostridial binding domain at the
amino terminus of a modified Clostridial toxin; locating a
non-Clostridial binding domain between a Clostridial toxin
enzymatic domain and a translocation domain of a modified
Clostridial toxin; and locating a non-Clostridial binding domain at
the carboxyl terminus of a modified Clostridial toxin. Other
non-limiting examples include, locating a non-Clostridial binding
domain between a Clostridial toxin enzymatic domain and a
Clostridial toxin translocation domain of a modified Clostridial
toxin. The enzymatic domain of naturally-occurring Clostridial
toxins contains the native start methionine. Thus, in domain
organizations where the enzymatic domain is not in the
amino-terminal location an amino acid sequence comprising the start
methionine should be placed in front of the amino-terminal domain.
Likewise, where a non-Clostridial binding domain is in the
amino-terminal position, an amino acid sequence comprising a start
methionine and a protease cleavage site may be operably-linked in
situations in which a non-Clostridial binding domain requires a
free amino terminus, see, e.g., Shengwen Li et al., Degradable
Clostridial Toxins, U.S. patent application Ser. No. 11/572,512
(Jan. 23, 2007), which is hereby incorporated by reference in its
entirety. In addition, it is known in the art that when adding a
polypeptide that is operably-linked to the amino terminus of
another polypeptide comprising the start methionine that the
original methionine residue can be deleted.
[0230] Thus, in an embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order
comprising a non-Clostridial binding domain, a translocation
domain, an exogenous protease cleavage site and an enzymatic domain
(FIG. 3A). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a non-Clostridial binding domain, a Clostridial
toxin translocation domain, an exogenous protease cleavage site and
a Clostridial toxin enzymatic domain.
[0231] In another embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order
comprising a non-Clostridial binding domain, an enzymatic domain,
an exogenous protease cleavage site, and a translocation domain
(FIG. 3B). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a non-Clostridial binding domain, a Clostridial
toxin enzymatic domain, an exogenous protease cleavage site, a
Clostridial toxin translocation domain.
[0232] In yet another embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order
comprising an enzymatic domain, an exogenous protease cleavage
site, a non-Clostridial binding domain, and a translocation domain
(FIG. 4A). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a Clostridial toxin enzymatic domain, an exogenous
protease cleavage site, a non-Clostridial binding domain, and a
Clostridial toxin translocation domain.
[0233] In yet another embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order
comprising a translocation domain, an exogenous protease cleavage
site, a non-Clostridial binding domain, and an enzymatic domain
(FIG. 4B). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a Clostridial toxin translocation domain, a
non-Clostridial binding domain, an exogenous protease cleavage site
and a Clostridial toxin enzymatic domain.
[0234] In another embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order
comprising an enzymatic domain, a non-Clostridial binding domain,
an exogenous protease cleavage site, and a translocation domain
(FIG. 4C). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a Clostridial toxin enzymatic domain, a
non-Clostridial binding domain, an exogenous protease cleavage
site, a Clostridial toxin translocation domain.
[0235] In yet another embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order
comprising a translocation domain, a non-Clostridial binding
domain, an exogenous protease cleavage site and an enzymatic domain
(FIG. 4D). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a Clostridial toxin translocation domain, a
non-Clostridial binding domain, an exogenous protease cleavage site
and a Clostridial toxin enzymatic domain.
[0236] In still another embodiment, a modified Clostridial toxin
can comprise an amino to carboxyl single polypeptide linear order
comprising an enzymatic domain, an exogenous protease cleavage
site, a translocation domain, and a non-Clostridial binding domain
(FIG. 5A). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a Clostridial toxin enzymatic domain, an exogenous
protease cleavage site, a Clostridial toxin translocation domain,
and a non-Clostridial binding domain.
[0237] In still another embodiment, a modified Clostridial toxin
can comprise an amino to carboxyl single polypeptide linear order
comprising a translocation domain, an exogenous protease cleavage
site, an enzymatic domain and a non-Clostridial binding domain,
(FIG. 5B). In an aspect of this embodiment, a modified Clostridial
toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a Clostridial toxin translocation domain, a
non-Clostridial binding domain, an exogenous protease cleavage site
and a Clostridial toxin enzymatic domain.
[0238] Aspects of the present specification provide, in part, an
inactivation cleavage site. As used herein, the term "inactivation
cleavage site" refers to a scissile bond together with adjacent or
non-adjacent recognition elements, or both, sufficient for
selective proteolysis at the scissile bond by a protease present in
interstitial fluids or circulatory systems, such as, e.g., the
cardiovascular system or the lymphatic system. Such an inactivation
cleavage site is operably-linked as a fusion protein to a
Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification. By definition, an inactivation cleavage site
is susceptible to selective cleavage by at least one protease
present in interstitial fluids or circulatory systems. Non-limiting
examples of inactivation cleavage sites include Thrombin cleavage
sites, Plasmin cleavage sites, Coagulation Factor VIIa cleavage
sites, Coagulation Factor IXa cleavage sites, Coagulation Factor Xa
cleavage sites, Coagulation Factor XIa cleavage sites, Coagulation
Factor XIIa cleavage sites, plasma kallikrein cleavage sites,
protease-activated G protein-coupled receptor-1 (PAR1) cleavage
sites, PAR 2 cleavage sites, PAR3 cleavage sites, PAR4 cleavage
sites, Matrix Metalloproteinase-2 (MMP-2) cleavage sites, Matrix
Metalloproteinase-9 (MMP-9) cleavage sites, Furin cleavage sites,
urokinase-type Plasminogen activator (uPA) cleavage sites,
tissue-type Plasminogen activator (tPA) cleavage sites,
Tryptase-.epsilon. cleavage sites, Mouse mast cell protease-7
(mMCP-7) cleavage sites, endothelin-converting enzyme-1 (ECE-1)
cleavage sites, Kell blood group cleavage sites, DPPIV cleavage
sites, ADAM metallopeptidase with thrombospondin type 1 motif-13
(ADAMTS13) cleavage sites, and Cathepsin L cleavage sites (Table
4).
TABLE-US-00004 TABLE 4 Inactivation Cleavage Sites Protease
Reference Cleavage Site Sequences SEQ ID NO: Thrombin LVPR*GS 114
LVPK*GS 115 FIPR*TF 116 VLPR*SF 117 IVPR*SF 118 IVPR*GY 119 VVPR*GV
120 VLPR*LI 121 VMPR*SL 122 MFPR*SL 123 Coagulation KLTR*AETV 125
Factor VIIa DFTR*VVGG 126 (FVIIA) LSPR*TFHP 127 LIQR*NLSP 128
MATR*KMHD 129 LGIR*SFRN 130 PQGR*IVGG 131 NLTR*IVGG 132 QVVR*IVGG
133 Coagulation PQGR*IVGG 135 Factor IXa PQLR*MKNN 136 (FIXa)
NLTR*IVGG 137 QVVR*IVGG 138 Coagulation IDGR* 140 Factor Xa IEGR*
141 (FXa) IDGR*SVGG 142 IDGR*TVGG 143 IDGR*IVGG 144 IEGR*SVGG 145
IEGR*TVGG 146 IEGR*IVGG 147 PQGR*IVGG 148 IEGR*TSED 149 IEGR*IVEG
150 IDGR*IVEG 151 FNPR*TFGS 152 FDER*TFGL 153 IDER*IVGG 154
FNEK*TFGL 155 Coagulation AFWK*TDAS 157 Factor XIa KLTR*AETV 158
(FXIa) KLTR*AETI 159 DFTR*VVGG 160 EFSR*VVGG 161 KLTR*AETV 162
DFTR*VVGG 163 IKPR*IVGG 164 DLHR*HIFW 165 KQLR*VVNG 166 Coagulation
PQGR*IVGG 168 Factor XIIa IKPR*IVGG 169 (FXIIa) SMTR*VVGG 170
TSTR*IVGG 171 PMKR*LTLG 172 Kallikrein 1 SMTR*VVGG 174 SPFR*SSDI
175 SLMK*RPPG 176 YDWR*TPYL 177 SPFR*SVQV 178 SPFR*TPYL 179
TFHK*AEYR 180 PRFK*IIGG 181 ISLM*KRPP 182 LEAR*SAYH 183 EAKR*SYHS
184 PNRW*STGA 185 EAFY*SQFG 186 NAAR*STGA 187 SSEW*SMPY 188
GTLF*RSGN 189 ARLY*SRGA 190 EASR*SATL 191 EASY*RRKQ 192 TIFY*RRGA
193 AAWY*RTSR 194 SFHY*RMVG 195 ASSY*RTSR 196 TRFY*SRGR 197
IKFF*SAQT 198 Protein C KKTR*NLKK 200 LDRR*GLQR 201 MATR*KMHD 202
RLKK*SQFL 203 PQLR*MKNN 204 VDQR*GNQI 205 IEPR*SPSQ 206 KKTR*SPKT
207 LDQR*GVQR 208 PDPR*SKNN 209 Plasminogen GEAR*GSVI 211 GHAR*LVHV
212 AEFR*HDSG 213 HHQK*LVFF 214 GSNK*GALL 215 RAQR*SAGA 216
AFWK*TDAS 217 MSMR*VRRH 218 RGVR*RTAS 219 RAAR*SQCT 220 PQSR*SVPP
221 PYLK*VFNP 222 LSFR*ARAY 223 PQLR*RGWR 224 EDNR*DSSM 225
LSFR*ARAY 226 FRAR*AYGF 227 YGFR*GPGP 228 ITFR*MNVA 229 THEK*GRQS
230 PRLK*ARAG 231 PKAK*SHAP 232 PSHK*EGPQ 233 LFEK*KVYL 234
ADGK*KPSS 235 PRFK*IIGG 236 PQFR*IKGG 237 PRCR*HRPH 238 KGYR*SQRG
239 DVAQ*FVLT 240 Matrix Metallo- QPVS*VKVG 242 proteinase-2
RGVG*IKST 243 (MMP-2) FVDC*LIEQ 244 VPAG*NWVL 245 YHAD*IYDK 246
RACR*LAKA 247 QGAY*QEAF 248 DVLS*LLEK 249 TLDD*LIMA 250 HISS*LIKL
251 DPNN*LLND 252 PVQP*QQSP 253 KPKT*ITGP 254 VVHP*LVLL 255
HPLV*LLSV 256 AVAL*LIGP 257 QPLQ*LLDA 258 YIQG*INLV 259 LPQE*IKAN
260 NISD*LTAA 261 KPRA*LTAL 262 APSW*LLTA 263 AVRW*LLTA 264
AVSW*LLTA 265 SLRR*LTAA 266 SLSR*LTAL 267 RYSS*LTAA 268 SLAY*YTAL
269 SLRY*YTAA 270 SPAY*YTAL 271 MHKA*LTAA 272 LRLA*ITAL 273 Matrix
Metallo- IPEN*FFGV 275 proteinase-9 MDIA*IHHP 276 (MMP-9) SPSR*LFDQ
277 SEMR*LEKD 278 FSVN*LDVK 279 RLFD*QFFG 280 FFGE*HLLE 281
GLSE*MRLE 282 SPEE*LKVK 283 DVIE*VHGK 284 EVHG*KHEE 285 DEHG*FISR
286 GEHL*LESD 287 FHRK*YRIP 288 GPRK*QVSG 289 LSPF*YLRP 290
PPSF*LRAP 291 NPLE*NSGF 292 VPYG*LGSP 293 PPLK*LMHS 294 GPEG*LRVG
295 FMKG*LSKA 296 VVTG*VTAV 297 AIIG*LMVG 298 SDLG*LTGI 299
VPYG*LGSP 300 GAAG*VKGD 301 GPTG*KQGD 302 GPSG*DQGA 303 GPSG*FPFP
304 GAPG*FPGP 305 GAPG*NRGF 306 GLRG*ERGE 307 GPPG*SQGN 308
GPAG*QQGA 309 GPPG*KDGT 310 GQPG*SPGS 311 GSPG*YQGP 312 GPVS*AVLT
313 GPLG*MLSQ 314 GPLG*MWAQ 315 GPQG*IFGQ 316 LPRS*AKEL 317
NSFG*LRFG 318 RAIH*INAE 319 Furin RPRR*AKRF 321 RKKR*GLYA 322
RERR*RKKR 323 RKKR*GLYA 324 RKKR*TTSA 325 RHKR*ETLK 326 RLKR*DVVT
327 RMKR*EDLN 328 RAKR*FASL 329 RKKR*FVSS 330 RTKR*FLSY 331
RRAR*SVDG 332 VFRR*DAHK 333 VFRR*EAHK 334 RVAR*DITM 335 RISR*SLPQ
336 RSRR*AATS 337 RAKR*SPKH 338 FWHR*GVIK 339 AKRR*TKRD 340
AKRR*AKRD 341 AKQR*AKRD 342 RDVR*GFAS 343 RKRR*SVNP 344 RQKR*FVLS
345 RSKR*SLSC 346 u-Plasminogen GSGK*SATL 348 Activator QRGR*SATL
349 (u-PA) RGSV*ILTV 350 PSSR*RRVN 351 CPGR*VVGG 352 PGAR*GRAF 353
SSSR*GPTH 354 VSNK*YFSN 355 NSGR*AVTY 356 TYSR*SRYL 357
NSGR*AVTY 358 PSGR*GRIL 359 AGSR*AVYY 360 TYGR*SRTN 361 NSSR*GVYL
362 PSSR*SVYN 363 ASGR*GRTY 364 TSSR*AVYL 365 NSGR*SRTL 366
VSGR*IRTG 367 SSGR*IRTV 368 t-Plasminogen NALR*YAPD 370 Activator
CPGR*VVGG 371 (t-PA) PQFR*IKGG 372 ALSR*MAVL 373 Tryptase- *RVVGGE
375 (Prosemin) *RIVGGE 376 *RIIGGE 377 *RVVGGD 378 *RIVGGD 379
*RIIGGD 380 *KVVGGE 381 *KIVGGE 382 *KIIGGE 383 *KVVGGD 384 *KIVGGD
385 *KIIGGD 386 Mouse mast cell LSSR*QSPG 388 protease-7 LQAR*GASL
389 (mMCP-7) LGPK*AITM 390 LGPR*SAVY 391 Endothelin- HQKL*VFFA 393
converting HHQK*LVFF 394 enzyme-1 KLVF*FAED 395 (ECE-1) DRVY*IHPF
396 YIHP*FHLV 397 YGLG*SPRS 398 TPEH*VVPY 399 DIiw*VNTP 400
DIIW*INTP 401 CHLD*IIWV 402 HLDI*IWVN 403 CVYF*CHLD 404 SCSS*LMDK
405 ECVY*FCHL 406 RSKR*CSCS 407 RSKR*ALEN 408 GFSP*FRSS 409
PRRP*YILP 410 KPQQ*FFGL 411 PQQF*FGLM 412 Kell blood-group
DIIW*VNTP 414 protein (KBGP) DIIW*INTP 415 Cathepsin L MFLE*AIPM
417 KVFQ*EPLF 418 ATLT*FDHS 419 PLFY*EAPR 420 TGLR*DPFN 421
KILH*LPTS 422 AHLK*NSQE 423 APLT*AEIQ 424 EALF*AERK 425 EPLA*AERK
426 GTFT*SDYS 427 KYLD*SRRA 428 QDFV*QWLM 429 KQLA*TKAA 430
STFE*ERSY 431 LRLE*WPYQ 432 RGFF*YTPK 433 GFFY*TPKA 434 HFFK*NIVT
435 RGLS*LSRF 436 QWLG*APVP 437 NMLK*RGLP 438 LSLA*HTHQ 439
TPFA*ATSS 440 KLLA*VSGP 441 QLFR*RAVL 442 PRFK*IIGG 443 PAR1
*SFLLRN 445 *SFFLRN 446 *SFFLKN 447 *TFLLRN 448 *GFPGKF 449 *GYPAKF
450 *GYPLKF 451 *GYPIKF 452 PAR2 *SLIGKV 454 *SLIGRL 455 PAR3
*TFRGAP 457 *SFNGGP 458 *SFNGNE 459 PAR4 *GYPGQV 461 *AYPGKF 462
*TYPGKF 463 *GYPGKY 464 *GYPGKW 465 *GYPGKK 466 *GYPGKF 467 *GYPGRF
468 *GYPGFK 469 *GYPAKF 470 *GFPGKF 471 *GFPGKP 472 *SYPGKF 473
*SYPAKF 474 *SYPGRF 475 *SYAGKF 476 *SFPGQP 477 *SFPGQA 478
ADAMTS13 NLVY*MVTG 479 An asterisks (*) indicates the peptide bond
of the P.sub.1-P.sub.1' cleavage site that is cleaved by the
indicated protease.
[0239] It is envisioned that an inactivation cleavage site of any
and all lengths can be useful in aspects of the present
specification with the proviso that the inactivation cleavage site
is capable of being cleaved by a interstitial fluid or circulatory
system protease. Thus, in aspects of this embodiment, an
inactivation cleavage site can be, e.g., at least 3, 4, 5, 6, 7, 8,
9, 10, 15, or 20 amino acids in length. In other aspects of this
embodiment, an inactivation cleavage site can be, e.g., at most 3,
4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acids in length.
[0240] An inactivation cleavage site useful in aspects of the
specification includes, without limitation, naturally occurring
inactivation cleavage site; naturally occurring inactivation
cleavage site variants; and non-naturally-occurring inactivation
cleavage site variants, such as, e.g., conservative inactivation
cleavage site variants, non-conservative inactivation cleavage site
variants and inactivation cleavage site peptidomimetics. As used
herein, the term "inactivation cleavage site variant," whether
naturally-occurring or non-naturally-occurring, refers to an
inactivation cleavage site that has at least one amino acid change
from the corresponding region of the disclosed reference sequences
and can be described in percent identity to the corresponding
region of that reference sequence. Any of a variety of sequence
alignment methods can be used to determine percent identity,
including, without limitation, global methods, local methods and
hybrid methods, such as, e.g., segment approach methods. Protocols
to determine percent identity are routine procedures within the
scope of one skilled in the art and from the teaching herein.
[0241] As used herein, the term "naturally occurring inactivation
cleavage site variant" refers to any inactivation cleavage site
produced without the aid of any human manipulation. Non-limiting
examples of inactivation cleavage sites include Thrombin cleavage
site variants, Plasmin cleavage site variants, Coagulation Factor V
cleavage site variants, Coagulation Factor VII cleavage site
variants, Coagulation Factor VIII cleavage site variants,
Coagulation Factor IXa cleavage site variants, Coagulation Factor
Xa cleavage site variants, Coagulation Factor XIa cleavage site
variants, Coagulation Factor XIIa cleavage site variants, plasma
kallikrein cleavage site variants, MMP-2 cleavage site variants,
MMP-9 cleavage site variants, Furin cleavage site variants,
u-Plasminogen activator cleavage site variants, t-Plasminogen
activator cleavage site variants, Tryptase-.epsilon. cleavage site
variants, mMCP-7 cleavage site variants, ECE-1 cleavage site
variants, KBGP cleavage site variants, Cathepsin L cleavage site
variants, PAR1 cleavage site variants, PAR2 cleavage site variants,
PAR3 cleavage site variants, PAR4 cleavage site variants, and
ADAM-TS13 cleavage site variants.
[0242] As used herein, the term "non-naturally occurring
inactivation cleavage site variant" refers to any inactivation
cleavage site produced with the aid of human manipulation,
including, without limitation, inactivation cleavage site variants
produced by genetic engineering using random mutagenesis or
rational design and inactivation cleavage site variants produced by
chemical synthesis. Non-limiting examples of non-naturally
occurring inactivation cleavage site variants include, e.g.,
conservative inactivation cleavage site variants, non-conservative
inactivation cleavage site variants, and inactivation cleavage site
peptidomimetics.
[0243] As used herein, the term "conservative inactivation cleavage
site variant" refers to an inactivation cleavage site that has at
least one amino acid substituted by another amino acid or an amino
acid analog that has at least one property similar to that of the
original amino acid from the reference inactivation cleavage site
sequence. Examples of properties include, without limitation,
similar size, topography, charge, hydrophobicity, hydrophilicity,
lipophilicity, covalent-bonding capacity, hydrogen-bonding
capacity, a physicochemical property, of the like, or any
combination thereof. A conservative inactivation cleavage site
variant can function in substantially the same manner as the
reference inactivation cleavage site on which the conservative
inactivation cleavage site variant is based, and can be substituted
for the reference inactivation cleavage site in any aspect of the
present specification. Non-limiting examples of a conservative
inactivation cleavage site variant include, e.g., conservative
Thrombin cleavage site variants, conservative Plasmin cleavage site
variants, conservative Coagulation Factor V cleavage site variants,
conservative Coagulation Factor VII cleavage site variants,
conservative Coagulation Factor VIII cleavage site variants,
conservative Coagulation Factor IXa cleavage site variants,
conservative Coagulation Factor Xa cleavage site variants,
conservative Coagulation Factor XIa cleavage site variants,
conservative Coagulation Factor XIIa cleavage site variants,
conservative plasma kallikrein cleavage site variants, conservative
MMP-2 cleavage site variants, conservative MMP-9 cleavage site
variants, conservative Furin cleavage site variants, conservative
u-Plasminogen activator cleavage site variants, conservative
t-Plasminogen activator cleavage site variants, conservative
Tryptase-.epsilon. cleavage site variants, conservative mMCP-7
cleavage site variants, conservative ECE-1 cleavage site variants,
conservative KBGP cleavage site variants, conservative Cathepsin L
cleavage site variants, conservative PAR1 cleavage site variants,
conservative PAR2 cleavage site variants, conservative PAR3
cleavage site variants, conservative PAR4 cleavage site variants,
and conservative ADAM-TS13 cleavage site variants.
[0244] As used herein, the term "non-conservative inactivation
cleavage site variant" refers to an inactivation cleavage site in
which 1) at least one amino acid is deleted from the reference
inactivation cleavage site on which the non-conservative
inactivation cleavage site variant is based; 2) at least one amino
acid added to the reference inactivation cleavage site on which the
non-conservative inactivation cleavage site is based; or 3) at
least one amino acid is substituted by another amino acid or an
amino acid analog that does not share any property similar to that
of the original amino acid from the reference inactivation cleavage
site sequence (Table 4). A non-conservative inactivation cleavage
site variant can function in substantially the same manner as the
reference inactivation cleavage site on which the non-conservative
inactivation cleavage site is based, and can be substituted for the
reference inactivation cleavage site in any aspect of the present
specification. Non-limiting examples of a non-conservative
inactivation cleavage site variant include, e.g., non-conservative
Thrombin cleavage site variants, non-conservative Plasmin cleavage
site variants, non-conservative Coagulation Factor V cleavage site
variants, non-conservative Coagulation Factor VII cleavage site
variants, non-conservative Coagulation Factor VIII cleavage site
variants, non-conservative Coagulation Factor IXa cleavage site
variants, non-conservative Coagulation Factor Xa cleavage site
variants, non-conservative Coagulation Factor XIa cleavage site
variants, non-conservative Coagulation Factor XIIa cleavage site
variants, non-conservative plasma kallikrein cleavage site
variants, non-conservative MMP-2 cleavage site variants,
non-conservative MMP-9 cleavage site variants, non-conservative
Furin cleavage site variants, non-conservative u-Plasminogen
activator cleavage site variants, non-conservative t-Plasminogen
activator cleavage site variants, non-conservative
Tryptase-.epsilon. cleavage site variants, non-conservative mMCP-7
cleavage site variants, non-conservative ECE-1 cleavage site
variants, non-conservative KBGP cleavage site variants,
non-conservative Cathepsin L cleavage site variants,
non-conservative PAR1 cleavage site variants, non-conservative PAR2
cleavage site variants, non-conservative PAR3 cleavage site
variants, non-conservative PAR4 cleavage site variants, and
non-conservative ADAM-TS13 cleavage site variants.
[0245] As used herein, the term "inactivation cleavage site
peptidomimetic" refers to an inactivation cleavage site that has at
least one amino acid substituted by a non-natural oligomer that has
at least one property similar to that of the first amino acid.
Examples of properties include, without limitation, topography of a
peptide primary structural element, functionality of a peptide
primary structural element, topology of a peptide secondary
structural element, functionality of a peptide secondary structural
element, of the like, or any combination thereof. An inactivation
cleavage site peptidomimetic can function in substantially the same
manner as the reference inactivation cleavage site on which the
inactivation cleavage site peptidomimetic is based, and can be
substituted for the reference inactivation cleavage site in any
aspect of the present specification. For examples of peptidomimetic
methods see, e.g., Amy S. Ripka & Daniel H. Rich,
Peptidomimetic design, 2(4) CURR. OPIN. CHEM. BIOL. 441-452 (1998);
and M. Angels Estiarte & Daniel H. Rich, Peptidomimetics for
Drug Design, 803-861 (BURGER'S MEDICINAL CHEMISTRY AND DRUG
DISCOVERY Vol. 1 PRINCIPLE AND PRACTICE, Donald J. Abraham ed.,
Wiley-Interscience, 6th ed 2003). Non-limiting examples of an
inactivation cleavage site peptidomimetic include, e.g., Thrombin
cleavage site peptidomimetics, Plasmin cleavage site
peptidomimetics, Coagulation Factor V cleavage site
peptidomimetics, Coagulation Factor VII cleavage site
peptidomimetics, Coagulation Factor VIII cleavage site
peptidomimetics, Coagulation Factor IXa cleavage site
peptidomimetics, Coagulation Factor Xa cleavage site
peptidomimetics, Coagulation Factor XIa cleavage site
peptidomimetics, Coagulation Factor XIIa cleavage site
peptidomimetics, plasma kallikrein cleavage site peptidomimetics,
MMP-2 cleavage site peptidomimetics, MMP-9 cleavage site
peptidomimetics, Furin cleavage site peptidomimetics, u-Plasminogen
activator cleavage site peptidomimetics, t-Plasminogen activator
cleavage site peptidomimetics, Tryptase-.epsilon. cleavage site
peptidomimetics, mMCP-7 cleavage site variants, ECE-1 cleavage site
peptidomimetics, KBGP cleavage site peptidomimetics, Cathepsin L
cleavage site peptidomimetics, PAR1 cleavage site peptidomimetics,
PAR2 cleavage site peptidomimetics, PAR3 cleavage site
peptidomimetics, PAR4 cleavage site peptidomimetics, and ADAM-TS13
cleavage site peptidomimetics.
[0246] Thus, in an embodiment, a Clostridial toxin comprises an
inactivation cleavage site. In an aspect of this embodiment, a
Clostridial toxin comprises a Clostridial toxin enzymatic domain, a
Clostridial toxin translocation domain, a Clostridial toxin binding
domain, and an inactivation cleavage site. In another aspect of
this embodiment, a Clostridial toxin comprises a naturally
occurring inactivation cleavage site variant, such as, e.g., an
inactivation cleavage site isoform. In another aspect of this
embodiment, a Clostridial toxin comprises a non-naturally occurring
inactivation cleavage site variant, such as, e.g., a conservative
inactivation cleavage site variant, a non-conservative inactivation
cleavage site variant or an active inactivation cleavage site
fragment, or any combination thereof.
[0247] In another embodiment, a Clostridial toxin chimeric
comprises an inactivation cleavage site. In an aspect of this
embodiment, a Clostridial toxin chimeric comprises a Clostridial
toxin enzymatic domain, a Clostridial toxin translocation domain, a
non-Clostridial toxin binding domain, and an inactivation cleavage
site. In another aspect of this embodiment, a Clostridial toxin
chimeric comprises a naturally occurring inactivation cleavage site
variant, such as, e.g., an inactivation cleavage site isoform. In
another aspect of this embodiment, a Clostridial toxin chimeric
comprises a non-naturally occurring inactivation cleavage site
variant, such as, e.g., a conservative inactivation cleavage site
variant, a non-conservative inactivation cleavage site variant or
an active inactivation cleavage site fragment, or any combination
thereof.
[0248] In another embodiment, a hydrophobic amino acid at one
particular position in the inactivation cleavage site can be
substituted with another hydrophobic amino acid. Examples of
hydrophobic amino acids include, e.g., C, F, I, L, M, V and W. In
another aspect of this embodiment, an aliphatic amino acid at one
particular position in the inactivation cleavage site can be
substituted with another aliphatic amino acid. Examples of
aliphatic amino acids include, e.g., A, I, L, P, and V. In yet
another aspect of this embodiment, an aromatic amino acid at one
particular position in the inactivation cleavage site can be
substituted with another aromatic amino acid. Examples of aromatic
amino acids include, e.g., F, H, W and Y. In still another aspect
of this embodiment, a stacking amino acid at one particular
position in the inactivation cleavage site can be substituted with
another stacking amino acid. Examples of stacking amino acids
include, e.g., F, H, W and Y. In a further aspect of this
embodiment, a polar amino acid at one particular position in the
inactivation cleavage site can be substituted with another polar
amino acid. Examples of polar amino acids include, e.g., D, E, K,
N, Q, and R. In a further aspect of this embodiment, a less polar
or indifferent amino acid at one particular position in the
inactivation cleavage site can be substituted with another less
polar or indifferent amino acid. Examples of less polar or
indifferent amino acids include, e.g., A, H, G, P, S, T, and Y. In
a yet further aspect of this embodiment, a positive charged amino
acid at one particular position in the inactivation cleavage site
can be substituted with another positive charged amino acid.
Examples of positive charged amino acids include, e.g., K, R, and
H. In a still further aspect of this embodiment, a negative charged
amino acid at one particular position in the inactivation cleavage
site can be substituted with another negative charged amino acid.
Examples of negative charged amino acids include, e.g., D and E. In
another aspect of this embodiment, a small amino acid at one
particular position in the inactivation cleavage site can be
substituted with another small amino acid. Examples of small amino
acids include, e.g., A, D, G, N, P, S, and T. In yet another aspect
of this embodiment, a C-beta branching amino acid at one particular
position in the inactivation cleavage site can be substituted with
another C-beta branching amino acid. Examples of C-beta branching
amino acids include, e.g., I, T and V.
[0249] Aspects of the present specification disclose, in part, a
Thrombin cleavage site as an inactivation cleavage site. As used
herein, the term "Thrombin cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
Thrombin under conditions suitable for Thrombin protease activity.
It is envisioned that any amino acid sequence cleaved by Thrombin
can be useful in aspects of the present specification. Although
exceptions are known, a generalized consensus sequence for a
Thrombin cleavage site is
X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.5X.sub.6X.sub.7 (SEQ ID NO:
113), where X.sub.1 is preferentially S, T, an amidic amino acid
like N and Q, a positive amino acid like H, K, and R, an aromatic
hydrophobic amino acid like F, W, and Y, an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.2 is any amino
acid; X.sub.3 is preferentially F, S, T, an amidic amino acid like
N or Q, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.4 is preferentially S, T, a positive amino acid
like H, K, and R, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M; and X.sub.5, X.sub.6, and X.sub.7, are
independently any amino acid. Table 4 lists exemplary reference
cleavage sites for Thrombin (SEQ ID NO: 114-123). Additional
Thrombin cleavage sites are well known in the art or can be defined
by routine methods. See, e.g., O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0250] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Thrombin cleavage site. In an aspect of
this embodiment, a Thrombin cleavage site comprises the consensus
sequence SEQ ID NO: 113, where X.sub.1 is S, T, an amidic amino
acid like N and Q, a positive amino acid like H, K, and R, an
aromatic hydrophobic amino acid like F, W, and Y, an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.2 is
any amino acid; X.sub.3 is F, S, T, an amidic amino acid like N or
Q, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.4 is S, T, a positive amino acid like H, K, and R, or
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
and X.sub.5, X.sub.6, and X.sub.7, are independently any amino
acid. In another aspect of this embodiment, a Thrombin cleavage
site comprises the consensus sequence SEQ ID NO: 113, where X.sub.1
is S, Q, K, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.2 is an acidic amino acid like D and E, an
amidic amino acid like N and Q, a basic amino acid like K and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is N, Q, G, P, A,
V, L, or I; X.sub.4 is S, T, H, G, A, L, or I; X.sub.5 is S, T, Q,
K, R, F, Y, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.6 is S, T, Q, K, R, G, P, A, V, L, or I; and
X.sub.7 is S, T, Q, K, R, G, P, A, V, L, or I. In another aspect of
this embodiment, a Thrombin cleavage site comprises the consensus
sequence SEQ ID NO: 113, where X.sub.1 is Q, G, P, A, V, L, I, or
M; X.sub.2 is S, T, D, E, G, A, V, or I; X.sub.3 is G, P, A, V, or
L; X.sub.4 is S, G, A, or L; X.sub.5 is Q, K, F, A, V, or L;
X.sub.6 is S, Q, K, R, G, P, V, or L; and X.sub.7 is S, T, K, G, V,
L, or I. In other aspects of this embodiment, a Thrombin cleavage
site comprises, e.g., SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:
116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:
120, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123.
[0251] Aspects of the present specification disclose, in part, a
Plasmin cleavage site as an inactivation cleavage site. As used
herein, the term "Plasmin cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
Plasmin under conditions suitable for Plasmin protease activity. It
is envisioned that any amino acid sequence cleaved by Plasmin can
be useful in aspects of the present specification. Plasmin
catalyzes cleavage of Lys| and Arg| bonds, with a specificity
similar to that of Trypsin. However, Plasmin is a much less
efficient enzyme than Trypsin, and cleaves only some of these bonds
in proteins. Trypsin cleaves peptide chains mainly at the carboxyl
side of the amino acids lysine or arginine, except when either is
followed by proline.
[0252] Aspects of the present specification disclose, in part, a
Coagulation Factor VIIa cleavage site as an inactivation cleavage
site. As used herein, the term "Coagulation Factor VIIa cleavage
site" or "FVIIa cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by FVIIa
under conditions suitable for FVIIa protease activity. It is
envisioned that any amino acid sequence cleaved by FVIIa can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a FVIIa cleavage
site is X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.5X.sub.6X.sub.7
(SEQ ID NO: 124), where X.sub.1 is preferentially an acidic amino
acid like D and E, an amidic amino acid like N and Q, a basic amino
acid like K and R, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M; X.sub.2 is Q, S, T, an aromatic hydrophobic
amino acid like F, W and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.3 is preferentially Q, S, T,
or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
M; X.sub.4, X.sub.5, X.sub.6, and X.sub.7, are independently any
amino acid. Table 4 lists exemplary reference cleavage sites for
FVIIa (SEQ ID NO: 125-133). Additional FVIIa cleavage sites are
well known in the art or can be defined by routine methods. See,
e.g., J. H. Morrissey, Coagulation Factor VIIa. In HANDBOOK OF
PROTEOLYTIC ENZYMES, pp. 1659-1662 (A. J. Barrett, N. D. Rawlings,
and J. F. Woessner, eds; Elsevier, London, 2d, 2004); O. Schilling
and C. M. Overall, Proteome-Derived, Database-Searchable Peptide
Libraries for Identifying Protease Cleavage Sites, Nat. Biotechnol.
26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0253] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Coagulation Factor VIIa cleavage site.
In an aspect of this embodiment, a Coagulation Factor VIIa cleavage
site comprises the consensus sequence SEQ ID NO: 124, where X.sub.1
is an acidic amino acid like D and E, an amidic amino acid like N
and Q, a basic amino acid like K and R, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.2 is Q, S, T, an
aromatic hydrophobic amino acid like F, W and Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.3 is Q,
S, T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; and X.sub.4, X.sub.5, X.sub.6, and X.sub.7, are
independently any amino acid. In other aspects of this embodiment,
a Coagulation Factor VIIa cleavage site comprises, e.g., SEQ ID NO:
125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:
129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, or SEQ ID NO:
133
[0254] Aspects of the present specification disclose, in part, a
Coagulation Factor IXa cleavage site as an inactivation cleavage
site. As used herein, the term "Coagulation Factor IXa cleavage
site" or "FIXa cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by FIXa
under conditions suitable for FIXa protease activity. It is
envisioned that any amino acid sequence cleaved by FIXa can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a FIXa cleavage
site is X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.5X.sub.6X.sub.7
(SEQ ID NO: 134), where X.sub.1 is preferentially an acidic amino
acid like D and E, an amidic amino acid like N and Q, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.2 is preferentially an acidic amino acid like D and E, an
amidic amino acid like N and Q, or an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and M; X.sub.3 is preferentially, S,
T, an aromatic hydrophobic amino acid like F, W and Y, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; and
X.sub.4, X.sub.5, X.sub.6, and X.sub.7, are independently any amino
acid. Table 4 lists exemplary reference cleavage sites for FIXa
(SEQ ID NO: 135-138). Additional FIXa cleavage sites are well known
in the art or can be defined by routine methods. See, e.g., A. T.
Thompson, Molecular Biology of Factor IX. In HEOSTASIS AND
THROMBOSIS, BASIC PRINCIPLES AND CLINICAL PRACTICE, pp. 128-129 (R.
W. Colman, J. Hirsh, V. J. Marder, A. W Clowes, J. N. George, eds;
Lippincott Williams & Wilkins, Philadelphia, Pa., 2d, 2001); S.
Kawabata and S. Iwanaga, Russellysin. In HANDBOOK OF PROTEOLYTIC
ENZYMES, pp. 683-684 (A. J. Barrett, N. D. Rawlings, and J. F.
Woessner, eds; Elsevier, London, 2d, 2004); A. E. Schmidt and S. P.
Bajaj, Coagulation factor IXa. In HANDBOOK OF PROTEOLYTIC ENZYMES,
pp. 1655-1659 (A. J. Barrett, N. D. Rawlings, and J. F. Woessner,
eds; Elsevier, London, 2d, 2004); O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0255] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Coagulation Factor IXa cleavage site. In
an aspect of this embodiment, a Coagulation Factor IXa cleavage
site comprises the consensus sequence SEQ ID NO: 134, where X.sub.1
is an acidic amino acid like D and E, an amidic amino acid like N
and Q, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.2 is an acidic amino acid like D and E, an amidic
amino acid like N and Q, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.3 is, S, T, an aromatic
hydrophobic amino acid like F, W and Y, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; and X.sub.4, X.sub.5,
X.sub.6, and X.sub.7, are independently any amino acid. In another
aspect of this embodiment, a Coagulation Factor IXa cleavage site
comprises the consensus sequence SEQ ID NO: 134, where X.sub.1 is
an acidic amino acid like D and E, an amidic amino acid like N and
Q, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.2 is an acidic amino acid like D and E, an amidic
amino acid like N and Q, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.3 is, S, T, an aromatic
hydrophobic amino acid like F, W and Y, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.4, X.sub.5,
X.sub.6, and X.sub.7, are independently an acidic amino acid like D
and E, an amidic amino acid like N and Q, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. In other
aspects of this embodiment, a Coagulation Factor IXa cleavage site
comprises, e.g., SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or
SEQ ID NO: 138.
[0256] Aspects of the present specification disclose, in part, a
Coagulation Factor Xa cleavage site as an inactivation cleavage
site. As used herein, the term "Coagulation Factor Xa cleavage
site" or "FXa cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by FXa
under conditions suitable for FXa protease activity. It is
envisioned that any amino acid sequence cleaved by FXa can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a FXa cleavage site
is X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.5X.sub.6X.sub.7 (SEQ ID
NO: 139), where X.sub.1 is any amino acid, X.sub.2 is
preferentially G, A, S, an acidic amino acid like D and E, an
amidic amino acid like Q and N, or an aromatic hydrophobic amino
acid like F, W and Y, X.sub.3 is preferentially an aromatic
hydrophobic amino acid like F, W and Y, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.4 is preferentially
an amidic amino acid like N and Q, an uncharged amino acid like C,
S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V,
L, I, and M; X.sub.5, X.sub.6, and X.sub.7, are independently any
amino acid. Table 4 lists exemplary reference cleavage sites for
FXa (SEQ ID NO: 140-155). Additional FXa cleavage sites are well
known in the art or can be defined by routine methods. See, e.g.,
D. L. Greenberg and E. W. Davie, Blood Coagulation Factors: Their
Complementary DNAs, Genes, and Expression. In HEMOSTASIS AND
THROMBOSIS, BASIC PRINCIPLES AND CLINICAL PRACTICE, pp. 34-35 (R.
W. Colman, J. Hirsh, V. J. Marder, A. W Clowes, J. N. George, eds;
Lippincott Williams & Wilkins, Philadelphia, Pa., 2d, 2001); O.
Schilling and C. M. Overall, Proteome-Derived, Database-Searchable
Peptide Libraries for Identifying Protease Cleavage Sites, Nat.
Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS:
The Peptidase Database, Nucleic Acids Res. 36(Database issue):
D320-D325 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 38(Database issue): D227-D233 (2010);
Neil D. Rawlings, et al., A Large and Accurate Collection of
Peptidase Cleavages in the MEROPS Database, Database in press
(2010), each of which is incorporated by reference in its
entirety.
[0257] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Coagulation Factor Xa cleavage site. In
an aspect of this embodiment, a Coagulation Factor Xa cleavage site
comprises the consensus sequence SEQ ID NO: 139, where X.sub.1 is
any amino acid, X.sub.2 is G, A, S, an acidic amino acid like D and
E, an amidic amino acid like Q and N, or an aromatic hydrophobic
amino acid like F, W and Y, X.sub.3 is an aromatic hydrophobic
amino acid like F, W and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.4 is an amidic amino acid like
N and Q, an uncharged amino acid like C, S, and T, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.5,
X.sub.6, and X.sub.7, are independently any amino acid. In another
aspect of this embodiment, a Coagulation Factor Xa cleavage site
comprises the consensus sequence SEQ ID NO: 139, where X.sub.1 is
E, F, P, A, L, or I; X.sub.2 is S, Q, D, E, F, G, or A; X.sub.3 is
F, G, or P; X.sub.4 is S, T, L, or I; X.sub.5 is S, F, A, or V;
X.sub.6 is S, T, E, N, H, G, A, or M; and X.sub.7 is S, N, D, Q, K,
R, or G. In another aspect of this embodiment, a Coagulation Factor
Xa cleavage site comprises the consensus sequence SEQ ID NO: 139,
where X.sub.1 is I or A; X.sub.2 is E or F; X.sub.3 is F, G, or P;
X.sub.4 is S, T, or I; X.sub.5 is S, F, or V; X.sub.6 is E or G;
and X.sub.7 is S or G. In other aspects of this embodiment, a
Coagulation Factor Xa cleavage site comprises, e.g., the amino acid
sequence SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:
144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO:
148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:
152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO: 155.
[0258] Aspects of the present specification disclose, in part, a
Coagulation Factor XIa cleavage site as an inactivation cleavage
site. As used herein, the term "Coagulation Factor XIa cleavage
site" or "FXIa cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by FXIa
under conditions suitable for FXIa protease activity. It is
envisioned that any amino acid sequence cleaved by FXIa can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a FXIa cleavage
site is X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.6X.sub.6X.sub.7
(SEQ ID NO: 156), where X.sub.1 is preferentially an acidic amino
acid like D or E, a basic amino acid like K and R, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.2 is
preferentially an acidic amino acid like D or E, an amidic amino
acid like Q and N, a basic amino acid like K and R, an aromatic
hydrophobic amino acid like F, W and Y, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is preferentially
H, an uncharged amino acid like C, S, and T, an aromatic
hydrophobic amino acid like F, W and Y, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.4 is preferentially
H, an uncharged amino acid like C, S, and T, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.5 is
preferentially an acidic amino acid like D and E, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.6 is
preferentially an amidic amino acid like Q and N, an uncharged
amino acid like C, S, and T, an aromatic hydrophobic amino acid
like F, W and Y, or an aliphatic hydrophobic amino acid like, G, P,
A, V, L, I, and M; and X.sub.7 is preferentially an uncharged amino
acid like C, S, and T, or an aliphatic hydrophobic amino acid like,
G, P, A, V, L, I, and M. Table 4 lists exemplary reference cleavage
sites for FXIa (SEQ ID NO: 157-166). Additional FXIa cleavage sites
are well known in the art or can be defined by routine methods.
See, e.g., P. N. Walsh, Coagulation Factor XIa. In Handbook of
Proteolytic Enzymes, pp. 1651-1655 (A. J. Barrett, N. D. Rawlings,
and J. F. Woessner, eds; Elsevier, London, 2d, 2004); O. Schilling
and C. M. Overall, Proteome-Derived, Database-Searchable Peptide
Libraries for Identifying Protease Cleavage Sites, Nat. Biotechnol.
26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0259] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Coagulation Factor XIa cleavage site. In
an aspect of this embodiment, a Coagulation Factor XIa cleavage
site comprises the consensus sequence SEQ ID NO: 156, where X.sub.1
is an acidic amino acid like D or E, a basic amino acid like K and
R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.2 is an acidic amino acid like D or E, an amidic amino
acid like Q and N, a basic amino acid like K and R, an aromatic
hydrophobic amino acid like F, W and Y, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is H, an
uncharged amino acid like C, S, and T, an aromatic hydrophobic
amino acid like F, W and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.4 is H, an uncharged amino
acid like C, S, and T, or an aliphatic hydrophobic amino acid like,
G, P, A, V, L, I, and M; X.sub.5 is an acidic amino acid like D and
E, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.6 is an amidic amino acid like Q and N, an uncharged
amino acid like C, S, and T, an aromatic hydrophobic amino acid
like F, Wand Y, or an aliphatic hydrophobic amino acid like, G, P,
A, V, L, I, and M; and X.sub.7 is an uncharged amino acid like C,
S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V,
L, I, and M. In another aspect of this embodiment, a Coagulation
Factor XIa cleavage site comprises the consensus sequence SEQ ID
NO: 156, where X.sub.1 is an acidic amino acid like D or E, or a
basic amino acid like K and R; X.sub.2 is an aromatic hydrophobic
amino acid like F, W and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.3 is an uncharged amino acid
like C, S, and T, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M; X.sub.4 is an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and M; X.sub.5 is an acidic amino acid
like D and E, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.6 is an uncharged amino acid like C, S, and
T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; and X.sub.7 is an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M. In another aspect of this embodiment, a
Coagulation Factor XIa cleavage site comprises the consensus
sequence SEQ ID NO: 156, where X.sub.1 is D or K; X.sub.2 is F or
L; X.sub.3 is T or P; X.sub.4 is A or V; X.sub.5 is E or V; X.sub.6
is T or G; and X.sub.7 is G or V. In other aspects of this
embodiment, a Coagulation Factor XIa cleavage site comprises, e.g.,
SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ
ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID
NO: 165, or SEQ ID NO: 166.
[0260] Aspects of the present specification disclose, in part, a
Coagulation Factor XIIa cleavage site as an inactivation cleavage
site. As used herein, the term "Coagulation Factor XIIa cleavage
site" or "FXIIa cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by FXIIa
under conditions suitable for FXIIa protease activity. It is
envisioned that any amino acid sequence cleaved by FXIIa can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a FXIIa cleavage
site is X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.6X.sub.6X.sub.7
(SEQ ID NO: 167), where X.sub.1 is preferentially an uncharged
amino acid like C, S, and T, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.2 is preferentially an acidic
amino acid like D and E, a basic amino acid like K and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is preferentially
a basic amino acid like K and R, an uncharged amino acid like C, S,
and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.4 is preferentially an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and M; X.sub.5 is preferentially an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.6 is preferentially
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
and X.sub.7 is preferentially an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M. Table 4 lists exemplary reference
cleavage sites for FXIIa (SEQ ID NO: 168-172). Additional FXIIa
cleavage sites are well known in the art or can be defined by
routine methods. See, e.g., O. D. Ratnoff, Coagulation Factor XIIa.
In Handbook of Proteolytic Enzymes, pp. 1642-1644 (A. J. Barrett,
N. D. Rawlings, and J. F. Woessner, eds; Elsevier, London, 2d,
2004); O. Schilling and C. M. Overall, Proteome-Derived,
Database-Searchable Peptide Libraries for Identifying Protease
Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 36(Database issue): D320-D325 (2008); Neil D. Rawlings, et
al., MEROPS: The Peptidase Database, Nucleic Acids Res. 38(Database
issue): D227-D233 (2010); Neil D. Rawlings, et al., A Large and
Accurate Collection of Peptidase Cleavages in the MEROPS Database,
Database in press (2010), each of which is incorporated by
reference in its entirety.
[0261] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Coagulation Factor XIIa cleavage site.
In an aspect of this embodiment, a Coagulation Factor XIIa cleavage
site comprises the consensus sequence SEQ ID NO: 167, where X.sub.1
is an uncharged amino acid like C, S, and T, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.2 is an
acidic amino acid like D and E, a basic amino acid like K and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is a basic amino
acid like K and R, an uncharged amino acid like C, S, and T, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.4 is an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.5 is an uncharged amino acid like C, S, and T, or
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.6 is an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; and X.sub.7 is an aliphatic hydrophobic amino acid like,
G, P, A, V, L, I, and M. In other aspect of this embodiment, a
Coagulation Factor XIIa cleavage site comprises the consensus
sequence SEQ ID NO: 167, where X.sub.1 is S, T, P, or I; X.sub.2 is
Q, K, S, or M; X.sub.3 is K, T, G, or P; X.sub.4 is L, I, or V;
X.sub.5 is T or V; X.sub.6 is G or L; and X.sub.7 is G. In other
aspects of this embodiment, a Coagulation Factor XIIa cleavage site
comprises, e.g., SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170,
SEQ ID NO: 171, or SEQ ID NO: 172.
[0262] Aspects of the present specification disclose, in part, a
Kallikrein 1 cleavage site as an inactivation cleavage site. As
used herein, the term "Kallikrein 1 cleavage site" refers to a
scissile bond together with adjacent or non-adjacent recognition
elements, or both, sufficient for detectable proteolysis at the
scissile bond by Kallikrein 1 under conditions suitable for
Kallikrein 1 protease activity. It is envisioned that any amino
acid sequence cleaved by Kallikrein 1 can be useful in aspects of
the present specification. Although exceptions are known, a
generalized consensus sequence for a Kallikrein 1 cleavage site is
X.sub.1X.sub.2X.sub.3X.sub.4*(R/K/S)X.sub.5X.sub.6X.sub.7 (SEQ ID
NO: 173), where X.sub.1 is preferentially an acidic amino acid like
D and E, an amidic amino acid like Q and N, an uncharged amino acid
like C, S, and T, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M; X.sub.2 is any amino acid; X.sub.3 is any
amino acid; X.sub.4 is preferentially a positive amino acid like H,
K, and R, a large non-polar amino acid like F, I, L, M and V, or an
aromatic hydrophobic amino acid like F, W and Y; X.sub.5 is any
amino acid; X.sub.6 is any amino acid; and X.sub.7 is any amino
acid. Table 4 lists exemplary reference cleavage sites for
Kallikrein 1 (SEQ ID NO: 174-198). Additional Kallikrein 1 cleavage
sites are well known in the art or can be defined by routine
methods. See, e.g., R. W. Colman, Contact Activation Pathway:
Inflammation, Fibrinolytic, Anticoagulant, Antiadhesive, and
Antiangiogenic Activities. In HEMOSTASIS AND THROMBOSIS, BASIC
PRINCIPLES AND CLINICAL PRACTICE, pp. 103-104 (R. W. Colman, J.
Hirsh, V. J. Marder, A. W Clowes, J. N. George, eds; Lippincott
Williams & Wilkins, Philadelphia, Pa., 2d, 2001); J. Chao,
Human Kallikrein 1, Tissue Kallikrein. In Handbook of Proteolytic
Enzymes, pp. 1577-1580 (A. J. Barrett, N. D. Rawlings, and J. F.
Woessner, eds; Elsevier, London, 2d, 2004); H. X. Li, et al.,
Substrate Specificity of Human Kallikreins 1 and 6 Determined by
Phage Display, Protein Sci. 17: 664-672 (2008); O. Schilling and C.
M. Overall, Proteome-Derived, Database-Searchable Peptide Libraries
for Identifying Protease Cleavage Sites, Nat. Biotechnol. 26:
685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0263] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Kallikrein 1 cleavage site. In an aspect
of this embodiment, a Kallikrein 1 cleavage site comprises the
consensus sequence SEQ ID NO: 173, where X.sub.1 is an acidic amino
acid like D and E, an amidic amino acid like Q and N, an uncharged
amino acid like C, S, and T, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.2 is any amino acid; X.sub.3
is any amino acid; X.sub.4 is a positive amino acid like H, K, and
R, a large non-polar amino acid like F, I, L, M and V, or an
aromatic hydrophobic amino acid like F, W and Y; X.sub.5 is any
amino acid; X.sub.6 is any amino acid; and X.sub.7 is any amino
acid. In another aspect of this embodiment, a Kallikrein 1 cleavage
site comprises the consensus sequence SEQ ID NO: 173, where X.sub.1
is D, S, T, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.2 is S, T, A, P, or V; X.sub.3 is S, F, or L;
X.sub.4 is R or an aromatic hydrophobic amino acid like F, W and Y;
X.sub.5 is R, S, T, or A; X.sub.6 is R, S, or G; and X.sub.7 is R,
G, or A. In other aspects of this embodiment, a Kallikrein 1
cleavage site comprises, e.g., SEQ ID NO: 174, SEQ ID NO: 175, SEQ
ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID
NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO:
184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO:
188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:
192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:
196, SEQ ID NO: 197, or SEQ ID NO: 198.
[0264] Aspects of the present specification disclose, in part, a
Protein C cleavage site as an inactivation cleavage site. As used
herein, the term "Protein C cleavage site" refers to a scissile
bond together with adjacent or non-adjacent recognition elements,
or both, sufficient for detectable proteolysis at the scissile bond
by Protein C under conditions suitable for Protein C protease
activity. It is envisioned that any amino acid sequence cleaved by
Protein C can be useful in aspects of the present specification.
Although exceptions are known, a generalized consensus sequence for
a Protein C cleavage site is
X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.6X.sub.6X.sub.7 (SEQ ID NO:
199), where X.sub.1 is preferentially a basic amino acid like K and
R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.2 is preferentially an acidic amino acid like D and E,
an amidic amino acid like Q and N, a basic amino acid like K and R,
or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
M; X.sub.3 is preferentially an amidic amino acid like Q and N, a
basic amino acid like K and R, an uncharged amino acid like C, S,
and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.4 is preferentially an amidic amino acid like Q and
N, a basic amino acid like K and R, an uncharged amino acid like C,
S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V,
L, I, and M; X.sub.5 is preferentially an amidic amino acid like Q
and N, a basic amino acid like K and R, an aromatic hydrophobic
amino acid like F, W and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.6 is preferentially an amidic
amino acid like Q and N, a positive amino acid like H, K, and R, an
uncharged amino acid like C, S, and T, or an aromatic hydrophobic
amino acid like F, W and Y; X.sub.7 is preferentially an acidic
amino acid like D and E, an amidic amino acid like Q and N, a basic
amino acid like K and R, an uncharged amino acid like C, S, and T,
or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
M. Table 4 lists exemplary reference cleavage sites for Protein C
(SEQ ID NO: 200-209). Additional Protein C cleavage sites are well
known in the art or can be defined by routine methods. See, e.g.,
L. Shen and B. Dahiback, Protein C. In Handbook of Proteolytic
Enzymes, pp. 1673-1677 (A. J. Barrett, N. D. Rawlings, and J. F.
Woessner, eds; Elsevier, London, 2d, 2004); O. Schilling and C. M.
Overall, Proteome-Derived, Database-Searchable Peptide Libraries
for Identifying Protease Cleavage Sites, Nat. Biotechnol. 26:
685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0265] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Protein C cleavage site. In an aspect of
this embodiment, a Protein C cleavage site comprises the consensus
sequence SEQ ID NO: 199, where X.sub.1 is a basic amino acid like K
and R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.2 is an acidic amino acid like D and E, an amidic
amino acid like Q and N, a basic amino acid like K and R, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.3 and X.sub.4 are independently an amidic amino acid like Q
and N, a basic amino acid like K and R, an uncharged amino acid
like C, S, and T, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M; X.sub.5 is an amidic amino acid like Q and N,
a basic amino acid like K and R, an aromatic hydrophobic amino acid
like F, W and Y, or an aliphatic hydrophobic amino acid like, G, P,
A, V, L, I, and M; X.sub.6 is an amidic amino acid like Q and N, a
positive amino acid like H, K, and R, an uncharged amino acid like
C, S, and T, or an aromatic hydrophobic amino acid like F, W and Y;
X.sub.7 is an acidic amino acid like D and E, an amidic amino acid
like Q and N, a basic amino acid like K and R, an uncharged amino
acid like C, S, and T, or an aliphatic hydrophobic amino acid like,
G, P, A, V, L, I, and M. In another aspect of this embodiment, a
Protein C cleavage site comprises the sequence SEQ ID NO: 199,
where X.sub.1 is K, R, or an aliphatic hydrophobic amino acid like,
G, P, A, V, L, I, and M; X.sub.2 is D, E, Q, N, or K; X.sub.3 is P,
L, T, Q, K, or R; X.sub.4 is G, I, S, N, or K; X.sub.5 is Q, N, K,
F, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.6 is F, S, N, Q, K, or H; X.sub.7 is L, I, T, K, D, E,
Q, or N. In other aspects of this embodiment, a Protein C cleavage
site comprises, e.g., SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:
202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:
206, SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID NO: 209.
[0266] Aspects of the present specification disclose, in part, a
Plasminogen cleavage site as an inactivation cleavage site. As used
herein, the term "Plasminogen cleavage site" refers to a scissile
bond together with adjacent or non-adjacent recognition elements,
or both, sufficient for detectable proteolysis at the scissile bond
by Plasminogen under conditions suitable for Plasminogen protease
activity. It is envisioned that any amino acid sequence cleaved by
Plasminogen can be useful in aspects of the present specification.
Although exceptions are known, a generalized consensus sequence for
a Plasminogen cleavage site is
X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4X.sub.6X.sub.6X.sub.7 (SEQ ID NO:
210), where X.sub.1 is preferentially a positive amino acid like H,
K and R, an uncharged amino acid like C, S, and T, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.2 is
preferentially an amidic amino acid like N and Q, a positive amino
acid like H, K and R, an uncharged amino acid like C, S, and T, or
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.3 is preferentially an amidic amino acid like N and Q, an
uncharged amino acid like C, S, and T, an aromatic amino acid like
F, W and Y, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.4 is preferentially a positive amino acid
like H, K and R, an uncharged amino acid like C, S, and T, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.5 is preferentially a positive amino acid like H, K and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.6 is any amino
acid; X.sub.7 is preferentially H, F, Y, R, an uncharged amino acid
like C, S, and T, an aliphatic hydrophobic amino acid like, G, P,
A, V, L, I, and M. Table 4 lists exemplary reference cleavage sites
for Plasminogen (SEQ ID NO: 211-240). Additional Plasminogen
cleavage sites are well known in the art or can be defined by
routine methods. See, e.g., O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0267] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Plasminogen cleavage site. In an aspect
of this embodiment, a Plasminogen cleavage site comprises the
consensus sequence SEQ ID NO: 211, where X.sub.1 is a positive
amino acid like H, K and R, an uncharged amino acid like C, S, and
T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.2 is an amidic amino acid like N and Q, a positive
amino acid like H, K and R, an uncharged amino acid like C, S, and
T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.3 is an amidic amino acid like N and Q, an uncharged
amino acid like C, S, and T, an aromatic amino acid like F, W and
Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.4 is a positive amino acid like H, K and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.5 is a positive
amino acid like H, K and R, an uncharged amino acid like C, S, and
T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.6 is any amino acid; X.sub.7 is H, F, Y, R, an
uncharged amino acid like C, S, and T, an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M. In another aspect of this
embodiment, a Plasminogen cleavage site comprises the sequence SEQ
ID NO: 211, where X.sub.1 is K, R, S, T, A, G, L, or P; X.sub.2 is
D, E, Q, N, K, R, S, T, A, G, I or L; X.sub.3 is N, Q, S, F, Y, A
or L; X.sub.4 is K, R, S, A, G, L, or V; X.sub.5 is K, R, N, S, F,
Y, A, I, L, P, or V; X.sub.6 is K, R, N, S, F, Y, A, G, L, P or V;
X.sub.7 is R, S, T, F, Y, A, G, I, L, or P. In other aspects of
this embodiment, a Plasminogen cleavage site comprises, e.g., SEQ
ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID
NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:
219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:
223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO:
227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO:
231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO:
235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO:
239, or SEQ ID NO: 240.
[0268] Aspects of the present specification disclose, in part, a
Matrix Metalloproteinase-2 cleavage site as an inactivation
cleavage site. As used herein, the term "Matrix Metalloproteinase-2
cleavage site" or "MMP-2 cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
MMP-2 under conditions suitable for MMP-2 protease activity. It is
envisioned that any amino acid sequence cleaved by MMP-2 can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a MMP-2 cleavage
site is
X.sub.1(P/NV/L/I)X.sub.2X.sub.3*(V/L/I/F/Q)X.sub.4X.sub.5X.sub.6
(SEQ ID NO: 241), where X.sub.1, X.sub.2, X.sub.3, X.sub.4,
X.sub.5, and X.sub.6 are any amino acid. Table 4 lists exemplary
reference cleavage sites for MMP-2 (SEQ ID NO: 242-273). Additional
MMP-2 cleavage sites are well known in the art or can be defined by
routine methods. See, e.g., O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0269] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Matrix Metalloproteinase-2 cleavage
site. In an aspect of this embodiment, a Matrix Metalloproteinase-2
cleavage site comprises the consensus sequence SEQ ID NO: 241,
where X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, and X.sub.6 are
any amino acid. In another aspect of this embodiment, a Matrix
Metalloproteinase-2 cleavage site comprises the consensus sequence
SEQ ID NO: 241, where X.sub.1 is an acidic amino acid like D and E,
an amidic amino acid like N and Q, an uncharged amino acid like C,
S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V,
L, I, and M; X.sub.2 is an acidic amino acid like D and E, a basic
amino acid like K and R, an amidic amino acid like N and Q, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is H, an acidic
amino acid like D and E, an amidic amino acid like N and Q, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.4 is a basic amino
acid like K and R, an amidic amino acid like N and Q, an uncharged
amino acid like C, S, and T, an aromatic amino acid like F, W, and
Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M; X.sub.5 is an acidic amino acid like D and E, a basic amino
acid like K and R, an amidic amino acid like N and Q, an uncharged
amino acid like C, S, and T, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.6 is an acidic amino acid like
D and E, a basic amino acid like K and R, an amidic amino acid like
N and Q, an uncharged amino acid like C, S, and T, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. In another
aspect of this embodiment, a Matrix Metalloproteinase-2 cleavage
site comprises the sequence SEQ ID NO: 241, where X.sub.1 is G, P,
A, V, L, I, S, T, E, or Q; X.sub.2 is G, A, L, S, N, Q, W, or K;
X.sub.3 is G, P, A, S, Q, D, E, or H; X.sub.4 is G, A, V, L, I, F,
S, T, Q, or K; X.sub.5 is G, A, V, S, T, Q, or K; X.sub.6 is G, P,
A, V, L, I, S, T, D, E, K, N, or Q. In another aspect of this
embodiment, a Matrix Metalloproteinase-2 cleavage site comprises
the sequence SEQ ID NO: 241, where X.sub.1 is G, A, or L, or Q;
X.sub.2 is G, A, or S; X.sub.3 is G, A, S, or N; X.sub.4 is A, V,
L, I, or K; X.sub.5 is G, A, or S; X.sub.6 is G, P, A, V, L, or D.
In other aspects of this embodiment, a Matrix Metalloproteinase-2
cleavage site comprises, e.g., SEQ ID NO: 242, SEQ ID NO: 243, SEQ
ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID
NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO:
252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO:
256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO:
260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO:
264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO:
268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO:
272, or SEQ ID NO: 273.
[0270] Aspects of the present specification disclose, in part, a
Matrix Metalloproteinase-9 cleavage site as an inactivation
cleavage site. As used herein, the term "Matrix Metalloproteinase-9
cleavage site" or "MMP-2 cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
MMP-9 under conditions suitable for MMP-9 protease activity. It is
envisioned that any amino acid sequence cleaved by MMP-9 can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a MMP-9 cleavage
site is X.sub.1X.sub.2X.sub.3X.sub.4*X.sub.5X.sub.6X.sub.7X.sub.8
(SEQ ID NO: 274), where X.sub.1 is preferentially F, an acidic
amino acid like D and E, an amidic amino acid like N and Q, a
positive amino acid like H, K, and R, an uncharged amino acid like
C, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.2 is preferentially F, Y, S, T, an acidic
amino acid like D and E, an amidic amino acid like N and Q, a
positive amino acid like H, K, and R, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is preferentially
F, Y, an acidic amino acid like D and E, an amidic amino acid like
N and Q, a positive amino acid like H, K, and R, an uncharged amino
acid like C, S, and T, or an aliphatic hydrophobic amino acid like,
G, P, A, V, L, I, and M; X.sub.4 is any amino acid; X.sub.5 is
preferentially S, T, an acidic amino acid like D and E, an amidic
amino acid like N and Q, a positive amino acid like H, K, and R, an
aromatic hydrophobic amino acid like F, W, and Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.6 is
any amino acid; X.sub.7 is any amino acid; X.sub.8 is
preferentially F, Y, an acidic amino acid like D and E, an amidic
amino acid like N and Q, a positive amino acid like H, K, and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M. Table 4 lists exemplary
reference cleavage sites for MMP-9 (SEQ ID NO: 275-319). Additional
MMP-9 cleavage sites are well known in the art or can be defined by
routine methods. See, e.g., S. L. Kridel, et al., Substrate
Hydrolysis by Matrix Metalloproteinase-9, J. Biol. Chem. 276:
20572-20578 (2001); E. Y. Zhen, et al., Characterization of
Metalloprotease Cleavage Products of Human Articular Cartilage,
Arthritis Rheum. 58: 2420-2431 (2008); O. Schilling and C. M.
Overall, Proteome-Derived, Database-Searchable Peptide Libraries
for Identifying Protease Cleavage Sites, Nat. Biotechnol. 26:
685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0271] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Matrix Metalloproteinase-9 cleavage
site. In an aspect of this embodiment, a Matrix Metalloproteinase-9
cleavage site comprises the consensus sequence SEQ ID NO: 274,
where X.sub.1 is F, an acidic amino acid like D and E, an amidic
amino acid like N and Q, a positive amino acid like H, K, and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.2 is F, Y, S, T, an
acidic amino acid like D and E, an amidic amino acid like N and Q,
a positive amino acid like H, K, and R, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is F, Y, an
acidic amino acid like D and E, an amidic amino acid like N and Q,
a positive amino acid like H, K, and R, an uncharged amino acid
like C, S, and T, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M; X.sub.4 is any amino acid; X.sub.5 is S, T,
an acidic amino acid like D and E, an amidic amino acid like N and
Q, a positive amino acid like H, K, and R, an aromatic hydrophobic
amino acid like F, W, and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; X.sub.6 is any amino acid; X.sub.7
is any amino acid; X.sub.8 is F, Y, an acidic amino acid like D and
E, an amidic amino acid like N and Q, a positive amino acid like H,
K, and R, an uncharged amino acid like C, S, and T, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. In another
aspect of this embodiment, a Matrix Metalloproteinase-9 cleavage
site comprises the consensus sequence SEQ ID NO: 274, where X.sub.1
is G, V, L, I, F, S, Q, K, or R; X.sub.2 is P, A, V, L, I, or S;
X.sub.3 is G, P, A, V, L, S, Q, E, K, or R; X.sub.4 is G, P, A, V,
L, F, S, N, E, or K; X.sub.5 is A, V, L, I, M, F, S, Q, or K;
X.sub.6 is P, A, V, L, I, S, T, Q, E, K, or R; X.sub.7 is G, A, V,
L, S, or T; X.sub.8 is G, P, A, V, L, F, T, D, E, K, or R. In
another aspect of this embodiment, a Matrix Metalloproteinase-9
cleavage site comprises the consensus sequence SEQ ID NO: 274,
where X.sub.1 is G or L; X.sub.2 is P, A, or V; X.sub.3 is P, A, R,
K, or S; X.sub.4 is G; X.sub.5 is A, V, L, or I; X.sub.6 is T, Q,
K, or R; X.sub.7 is G, A, or S; X.sub.8 is G, P, A, V, or E. In
other aspects of this embodiment, a Matrix Metalloproteinase-9
cleavage site comprises, e.g., SEQ ID NO: 275, SEQ ID NO: 276, SEQ
ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID
NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO:
285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO:
289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO:
293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO:
297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO:
301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO:
305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO:
309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO:
313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO:
317, SEQ ID NO: 318, or SEQ ID NO: 319.
[0272] Aspects of the present specification disclose, in part, a
Furin cleavage site as an inactivation cleavage site. As used
herein, the term "Furin cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
Furin under conditions suitable for Furin protease activity. It is
envisioned that any amino acid sequence cleaved by Furin can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a Furin cleavage
site is (R/I/A)X.sub.1(R/K/A/P)R*X.sub.2*X.sub.3X.sub.4X.sub.5 (SEQ
ID NO: 320), where X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5
are any amino acid. Table 4 lists exemplary reference cleavage
sites for Furin (SEQ ID NO: 321-346). Additional Furin cleavage
sites are well known in the art or can be defined by routine
methods. See, e.g., A. Basak, et al., Implication of the Proprotein
Convertases Furin, PC5 And PC7 in the Cleavage of Surface
Glycoproteins of Hong Kong, Ebola and Respiratory Syncytial
Viruses: A Comparative Analysis with Fluorogenic Peptides, Biochem.
J. 353: 537-545 (2001); O. Bader, et al., Processing of Predicted
Substrates of Fungal Kex2 Proteinases from Candida albicans, C.
glabrata, Saccharomyces cerevisiae and Pichia pastoris, BMC
Microbiol. 8: 116 (2008); O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0273] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Furin cleavage site. In an aspect of
this embodiment, a Furin cleavage site comprises the consensus
sequence SEQ ID NO: 320, where X.sub.1, X.sub.2, X.sub.3, X.sub.4,
and X.sub.5 are any amino acid. In another aspect of this
embodiment, a Furin cleavage site comprises the consensus sequence
SEQ ID NO: 320, where X.sub.1 is F, S, T, an acidic amino acid like
D and E, an amidic amino acid like N and Q, a positive amino acid
like H, K, and R, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M; X.sub.2 is G, P, M, F, Y, an acidic amino
acid like D and E, an amidic amino acid like N and Q, a positive
amino acid like H, K, and R, or an uncharged amino acid like C, S,
and T; X.sub.3 is G, P, A, V, L, I, F, W, S, T, N, Q, D, H, K, or
R; X.sub.4 is F, Y, an acidic amino acid like D and E, an amidic
amino acid like N and Q, a positive amino acid like H, K, and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; and X.sub.5 is F, Y, an
acidic amino acid like D and E, an amidic amino acid like N and Q,
a positive amino acid like H, K, and R, an uncharged amino acid
like C, S, and T, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and M. In another aspect of this embodiment, a Furin
cleavage site comprises the consensus sequence SEQ ID NO: 320,
where X.sub.1 is K, R, S or T; X.sub.2 is D, E, S, A or G; X.sub.3
is A, V, L, or I; and X.sub.4 is S, G, D, E or R; and X.sub.5 is G,
P, A, S, T, Q, D, or E. In other aspects of this embodiment, a
Furin cleavage site comprises, e.g., SEQ ID NO: 321, SEQ ID NO:
322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO:
326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO:
330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO:
334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO:
338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO:
342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, or SEQ ID NO:
346.
[0274] Aspects of the present specification disclose, in part, a
u-Plasminogen Activator cleavage site as an inactivation cleavage
site. As used herein, the term "u-Plasminogen Activator cleavage
site" or "u-PA cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by u-PA
under conditions suitable for u-PA protease activity. It is
envisioned that any amino acid sequence cleaved by u-Plasminogen
Activator can be useful in aspects of the present specification.
Although exceptions are known, a generalized consensus sequence for
a u-PA cleavage site is
X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4*X.sub.6X.sub.6X.sub.7 (SEQ ID
NO: 347), where X.sub.1 is any amino acid; X.sub.2 is
preferentially an uncharged amino acid like C, S, and T, an
aromatic amino acid like F, W, and Y, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; X.sub.3 is preferentially
an amidic amino acid like N and Q, an uncharged amino acid like C,
S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V,
L, I, and M; X.sub.4 is any amino acid; X.sub.5 is preferentially a
basic amino acid like K and R, an aromatic amino acid like F, W,
and Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.6 is preferentially an uncharged amino acid like C,
S, and T, an aromatic amino acid like F, W, and Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; and X.sub.7
is any amino acid. Table 4 lists exemplary reference cleavage sites
for u-PA (SEQ ID NO: 347-368). Additional u-PA cleavage sites are
well known in the art or can be defined by routine methods. See,
e.g., V. Ellis, u-Plasminogen Activator. In Handbook of Proteolytic
Enzymes, pp. 1677-1683 (A. J. Barrett, N. D. Rawlings, and J. F.
Woessner, eds; Elsevier, London, 2d, 2004); O. Schilling and C. M.
Overall, Proteome-Derived, Database-Searchable Peptide Libraries
for Identifying Protease Cleavage Sites, Nat. Biotechnol. 26:
685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0275] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a u-Plasminogen Activator cleavage site.
In an aspect of this embodiment, a u-Plasminogen Activator cleavage
site comprises the consensus sequence SEQ ID NO: 347, where X.sub.1
is any amino acid; X.sub.2 is an uncharged amino acid like C, S,
and T, an aromatic amino acid like F, W, and Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.3 is an
amidic amino acid like N and Q, an uncharged amino acid like C, S,
and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.4 is any amino acid; X.sub.5 is a basic amino acid
like K and R, an aromatic amino acid like F, W, and Y, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.6 is an uncharged amino acid like C, S, and T, an aromatic
amino acid like F, W, and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; and X.sub.7 is any amino acid. In
another aspect of this embodiment, a u-Plasminogen Activator
cleavage site comprises the consensus sequence SEQ ID NO: 347,
where X.sub.1 is P, A, L, S, T, C, N, or R; X.sub.2 is G, P, L, Y,
S, or T; X.sub.3 is G, A, S, or N; and X.sub.4 is G, A, V, I, Y, S,
or R; X.sub.5 is P, V, L, F, or R; X.sub.6 is G, A, V, Y, S, or T;
and X.sub.7 is G, V, L, F, Y, N, or H. In another aspect of this
embodiment, a u-Plasminogen Activator cleavage site comprises the
consensus sequence SEQ ID NO: 347, where X.sub.1 is P, A, L, S, T,
C, N, or R; X.sub.2 is G, Y, or S; X.sub.3 is G or S; and X.sub.4
is G, A, V, I, Y, S, or R; X.sub.4 is V or R; X.sub.6 is T or Y;
and X.sub.7 is G, V, L, F, Y, N, or H. In other aspects of this
embodiment, a u-Plasminogen Activator cleavage site comprises,
e.g., SEQ ID NO: 348, SEQ ID NO: 349, NO: 350, SEQ ID NO: 351, SEQ
ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, or SEQ
ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID
NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO:
364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, or SEQ ID NO:
368.
[0276] Aspects of the present specification disclose, in part, a
t-Plasminogen Activator cleavage site as an inactivation cleavage
site. As used herein, the term "t-Plasminogen Activator cleavage
site" or "t-PA cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by t-PA
under conditions suitable for t-PA protease activity. It is
envisioned that any amino acid sequence cleaved by t-PA can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a t-PA cleavage
site is X.sub.1X.sub.2X.sub.3(R/K)*X.sub.4*X.sub.5X.sub.6X.sub.7
(SEQ ID NO: 369), where X.sub.1, X.sub.2, X.sub.3, X.sub.4,
X.sub.5, X.sub.6, and X.sub.7 are any amino acid. Table 4 lists
exemplary reference cleavage sites for t-PA (SEQ ID NO: 370-373).
Additional t-PA cleavage sites are well known in the art or can be
defined by routine methods. See, e.g., H. R. Lijnen and D. Collen,
t-Plasminogen Activator. In Handbook of Proteolytic Enzymes, pp.
1684-1689 (A. J. Barrett, N. D. Rawlings, and J. F. Woessner, eds;
Elsevier, London, 2d, 2004); O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0277] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a t-Plasminogen Activator cleavage site.
In an aspect of this embodiment, a t-Plasminogen Activator cleavage
site comprises the consensus sequence SEQ ID NO: 369, where
X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, and X.sub.7
are any amino acid. In another aspect of this embodiment, a
t-Plasminogen Activator cleavage site comprises the consensus
sequence SEQ ID NO: 369, where X.sub.1 is an amidic amino acid like
N and Q, an uncharged amino acid like C, S, and T, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.2 is an
amidic amino acid like N and Q, or an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and M; X.sub.3 is an amidic amino acid
like N and Q, an aromatic hydrophobic amino acid like F, W and Y,
or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
M; X.sub.4 is an aromatic hydrophobic amino acid like F, W and Y,
or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
M; X.sub.5 is a basic amino acid like K and R, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.6 is an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; and
X.sub.7 is an acidic amino acid like D and E, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. In another
aspect of this embodiment, a t-Plasminogen Activator cleavage site
comprises the consensus sequence SEQ ID NO: 369, where X.sub.1 is
A, P, C, or N; X.sub.2 is A, L, P, or Q; X.sub.3 is G, L, S, or F;
X.sub.4 is I, V, M, or Y; X.sub.5 is A, V, or K; X.sub.6 is G, V,
or P; and X.sub.7 is G, L, or D. In other aspects of this
embodiment, a t-Plasminogen Activator cleavage site comprises,
e.g., SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, or SEQ ID NO:
373.
[0278] Aspects of the present specification disclose, in part, a
Tryptase-.epsilon. cleavage site as an inactivation cleavage site.
As used herein, the term "Tryptase-.epsilon. cleavage site" or
"Prosemin cleavage site" refers to a scissile bond together with
adjacent or non-adjacent recognition elements, or both, sufficient
for detectable proteolysis at the scissile bond by
Tryptase-.epsilon. under conditions suitable for Tryptase-.epsilon.
protease activity. It is envisioned that any amino acid sequence
cleaved by Tryptase-.epsilon. can be useful in aspects of the
present specification. Although exceptions are known, a generalized
consensus sequence for a Tryptase-.epsilon. cleavage site is
*(R/K)X.sub.1X.sub.2X.sub.3X.sub.4(D/E) (SEQ ID NO: 374), where
X.sub.1, X.sub.2, X.sub.3, and X.sub.4, are independently an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M.
Table 4 lists exemplary reference cleavage sites for
Tryptase-.epsilon. (SEQ ID NO: 375-386). Additional
Tryptase-.epsilon. cleavage sites are well known in the art or can
be defined by routine methods. See, e.g., O. Schilling and C. M.
Overall, Proteome-Derived, Database-Searchable Peptide Libraries
for Identifying Protease Cleavage Sites, Nat. Biotechnol. 26:
685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0279] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Tryptase-.epsilon. cleavage site. In an
aspect of this embodiment, a Tryptase-.epsilon. cleavage site
comprises the consensus sequence SEQ ID NO: 374, where X.sub.1,
X.sub.2, X.sub.3, and X.sub.4, are independently an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. In another
aspect of this embodiment, a Tryptase-.epsilon. cleavage site
comprises the consensus sequence SEQ ID NO: 374, where X.sub.1 is I
or V; X.sub.2 is I or V; X.sub.3 is G or S; X.sub.4 is G or S. In
other aspects of this embodiment, a Tryptase-.epsilon. cleavage
site comprises, e.g., SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO:
377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO:
381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO:
385, or SEQ ID NO: 386.
[0280] Aspects of the present specification disclose, in part, a
Mouse Mast Cell Protease-7 cleavage site as an inactivation
cleavage site. As used herein, the term "Mouse Mast Cell Protease-7
cleavage site" or "mMCP-7 cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
mMCP-7 under conditions suitable for mMCP-7 protease activity. It
is envisioned that any amino acid sequence cleaved by mMCP-7 can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a mMCP-7 cleavage
site is X.sub.1X.sub.2X.sub.3(K/R)*X.sub.4X.sub.5X.sub.6X.sub.7
(SEQ ID NO: 387), where X.sub.1 is any amino acid; X.sub.2 is
preferentially an amidic amino acid like N or Q, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.3 is
preferentially an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; and X.sub.4, X.sub.5, X.sub.6, X.sub.7 are any
amino acid. Table 4 lists exemplary reference cleavage sites for
mMMCP-7 (SEQ ID NO: 388-391). Additional mMMCP-7 cleavage sites are
well known in the art or can be defined by routine methods. See,
e.g., O. Schilling and C. M. Overall, Proteome-Derived,
Database-Searchable Peptide Libraries for Identifying Protease
Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 36(Database issue): D320-D325 (2008); Neil D. Rawlings, et
al., MEROPS: The Peptidase Database, Nucleic Acids Res. 38(Database
issue): D227-D233 (2010); Neil D. Rawlings, et al., A Large and
Accurate Collection of Peptidase Cleavages in the MEROPS Database,
Database in press (2010), each of which is incorporated by
reference in its entirety.
[0281] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Mouse Mast Cell Protease-7 cleavage
site. In an aspect of this embodiment, a Mouse Mast Cell Proteas-7
cleavage site comprises the consensus sequence SEQ ID NO: 387,
where X.sub.1 is any amino acid; X.sub.2 is an amidic amino acid
like N or Q, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.3 is an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M; and X.sub.4, X.sub.5, X.sub.6,
X.sub.7 are independently any amino acid. In another aspect of this
embodiment, a Mouse Mast Cell Protease-7 cleavage site comprises
the consensus sequence SEQ ID NO: 387, where X.sub.1 is any amino
acid; X.sub.2 is G, S, or Q; X.sub.3 is A, P or S; and X.sub.4,
X.sub.5, X.sub.6, X.sub.7 are any amino acid. In other aspects of
this embodiment, a Mouse Mast Cell Protease-7 cleavage site
comprises, e.g., SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, or
SEQ ID NO: 391.
[0282] Aspects of the present specification disclose, in part, an
Endothelin-Converting Enzyme-1 cleavage site as an inactivation
cleavage site. As used herein, the term "Endothelin-Converting
Enzyme-1 cleavage site" or "ECE-1 cleavage site" refers to a
scissile bond together with adjacent or non-adjacent recognition
elements, or both, sufficient for detectable proteolysis at the
scissile bond by ECE-1 under conditions suitable for ECE-1 protease
activity. It is envisioned that any amino acid sequence cleaved by
ECE-1 can be useful in aspects of the present specification.
Although exceptions are known, a generalized consensus sequence for
an ECE-1 cleavage site is
X.sub.1X.sub.2X.sub.3X.sub.4*(F/L/I/V/Y)X.sub.5X.sub.6X.sub.7 (SEQ
ID NO: 392), where X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5,
X.sub.6, and X.sub.7 are any amino acid. Table 4 lists exemplary
reference cleavage sites for ECE-1 (SEQ ID NO: 393-412). Additional
ECE-1 cleavage sites are well known in the art or can be defined by
routine methods. See, e.g., K. Ahn and G. D. Johnson,
Endothelin-Converting Enzyme-1. In Handbook of Proteolytic Enzymes,
pp. 429-434 (A. J. Barrett, N. D. Rawlings, and J. F. Woessner,
eds; Elsevier, London, 2d, 2004); O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0283] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises an Endothelin-Converting Enzyme-1 cleavage
site. In an aspect of this embodiment, an Endothelin-Converting
Enzyme-1 cleavage site comprises the consensus sequence SEQ ID NO:
392, where X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6,
and X.sub.7 are independently any amino acid. In another aspect of
this embodiment, an Endothelin-Converting Enzyme-1 cleavage site
comprises the consensus sequence SEQ ID NO: 392, where X.sub.1 is
G, P, Y, an acidic amino acid like D and E, an amidic amino acid
like N and Q, a positive amino acid like H, K, and R, or an
uncharged amino acid like C, S, and T; X.sub.2 is F, an acidic
amino acid like D and E, an amidic amino acid like N and Q, a
positive amino acid like H, K, and R, an uncharged amino acid like
C, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.3 is S, an acidic amino acid like D and E, an
amidic amino acid like N and Q, a positive amino acid like H, K,
and R, an aromatic hydrophobic amino acid like F, W and Y, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.4 is S, an acidic amino acid like D and E, an amidic amino
acid like N and Q, a positive amino acid like H, K, and R, an
aromatic hydrophobic amino acid like F, W and Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.5 is F,
W, S, C, N, E, a positive amino acid like H, K, and R, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.6 is G, P, V, L, F, Y, an acidic amino acid like D and E, an
amidic amino acid like N and Q, a positive amino acid like H, K,
and R, or an uncharged amino acid like C, S, and T; and X.sub.7 is
P, A, V, L, M, F, Y, S, N, D, or K. In another aspect of this
embodiment, an Endothelin-Converting Enzyme-1 cleavage site
comprises the consensus sequence SEQ ID NO: 392, where X.sub.1 is
G, P, Y, C, D, K, R, or H; X.sub.2 is P, L, I, F, S, C, Q, D, R, or
H; X.sub.3 is V, L, I, S, Q, K, or R; X.sub.4 is G, P, L, F, Y, W,
or R; X.sub.5 is V, I, M, F, N, R, or H; X.sub.6 is P, L, F, T, E,
or H; and X.sub.7 is P, V, L, F, S, N, D, or K. In another aspect
of this embodiment, an Endothelin-Converting Enzyme-1 cleavage site
comprises the consensus sequence SEQ ID NO: 392, where X.sub.1 is
G, D, or H; X.sub.2 is I or F; X.sub.3 is V, I, S, Q or K; X.sub.4
is P, F, or W; X.sub.5 is I, N, R, or H; X.sub.6 is L, T, or H; and
X.sub.7 is P, S, or D. In other aspects of this embodiment, an
Endothelin-Converting Enzyme-1 cleavage site comprises, e.g., SEQ
ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID
NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO:
401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO:
405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO:
409, SEQ ID NO: 410, SEQ ID NO: 411, or SEQ ID NO: 412.
[0284] Aspects of the present specification disclose, in part, a
Kell blood-group protein cleavage site as an inactivation cleavage
site. As used herein, the term "Kell blood-group protein cleavage
site" or KBGP cleavage site" refers to a scissile bond together
with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis at the scissile bond by KBGP
under conditions suitable for KBGP protease activity. It is
envisioned that any amino acid sequence cleaved by KBGP can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a KBGP cleavage
site is X.sub.1X.sub.2X.sub.3X.sub.4*X.sub.5X.sub.6X.sub.7X.sub.8
(SEQ ID NO: 413), where X.sub.1 is preferentially an acidic amino
acid like D and E; X.sub.2 is preferentially an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.3 is
preferentially an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.4 is preferentially an aromatic amino acid
like F, W, and Y; X.sub.5 is preferentially an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M; X.sub.6 is
preferentially an amidic amino acid like N and Q; X.sub.7 is an
uncharged amino acid like C, S, and T; X.sub.8 is preferentially an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M.
Table 4 lists exemplary reference cleavage sites for KBGP (SEQ ID
NO: 414-415). Additional KBGP cleavage sites are well known in the
art or can be defined by routine methods. See, e.g., O. Schilling
and C. M. Overall, Proteome-Derived, Database-Searchable Peptide
Libraries for Identifying Protease Cleavage Sites, Nat. Biotechnol.
26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: The Peptidase
Database, Nucleic Acids Res. 36(Database issue): D320-D325 (2008);
Neil D. Rawlings, et al., MEROPS: The Peptidase Database, Nucleic
Acids Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings,
et al., A Large and Accurate Collection of Peptidase Cleavages in
the MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0285] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Kell blood-group protein cleavage site.
In an aspect of this embodiment, a Kell blood-group protein
cleavage site comprises the consensus sequence SEQ ID NO: 413,
where X.sub.1 is an acidic amino acid like D and E; X.sub.2 is T or
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.3 is an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and M; X.sub.4 is an aromatic amino acid like F, W, and Y;
X.sub.5 is T or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.6 is an amidic amino acid like N and Q;
X.sub.7 is an uncharged amino acid like C, S, and T, or a C-beta
branched amino acid like I, V, or T; X.sub.8 is an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. In another
aspect of this embodiment, a Kell blood-group protein cleavage site
comprises the consensus sequence SEQ ID NO: 413, where X.sub.1 is
D; X.sub.2 is I, V, or T; X.sub.3 is I, V, or T; X.sub.4 is W;
X.sub.5 is I, V, or T; X.sub.6 is N; X.sub.7 is T; X.sub.8 is P. In
other aspects of this embodiment, a Kell blood-group protein
cleavage site comprises, e.g., SEQ ID NO: 414 or SEQ ID NO:
415.
[0286] Aspects of the present specification disclose, in part, a
Cathepsin L cleavage site as an inactivation cleavage site. As used
herein, the term "Cathepsin L cleavage site" refers to a scissile
bond together with adjacent or non-adjacent recognition elements,
or both, sufficient for detectable proteolysis at the scissile bond
by Cathepsin L under conditions suitable for Cathepsin L protease
activity. It is envisioned that any amino acid sequence cleaved by
Cathepsin L can be useful in aspects of the present specification.
Although exceptions are known, a generalized consensus sequence for
a Cathepsin L cleavage site is
X.sub.1X.sub.2X.sub.3X.sub.4*X.sub.5X.sub.6X.sub.7X.sub.8 (SEQ ID
NO: 416), where X.sub.1 is preferentially W, an acidic amino acid
like D and E, an amidic amino acid like N and Q, a positive amino
acid like H, K, and R, an uncharged amino acid like C, S, and T, or
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
X.sub.2 is any amino acid; X.sub.3 is preferentially L, V, F or Y;
and X.sub.4, X.sub.5, X.sub.6, X.sub.7, and X.sub.8 are any amino
acid. Table 4 lists exemplary reference cleavage sites for
Cathepsin L (SEQ ID NO: 417-443). Additional Cathepsin L cleavage
sites are well known in the art or can be defined by routine
methods. See, e.g., J. C. Kelly, et al., Profiling of Calpain
Activity with a Series of FRET-Based Substrates, Biochim. Biophys.
Acta 1794: 1505-1509 (2009); O. Schilling and C. M. Overall,
Proteome-Derived, Database-Searchable Peptide Libraries for
Identifying Protease Cleavage Sites, Nat. Biotechnol. 26: 685-694
(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,
Nucleic Acids Res. 36(Database issue): D320-D325 (2008); Neil D.
Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids
Res. 38(Database issue): D227-D233 (2010); Neil D. Rawlings, et
al., A Large and Accurate Collection of Peptidase Cleavages in the
MEROPS Database, Database in press (2010), each of which is
incorporated by reference in its entirety.
[0287] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a Cathepsin L cleavage site. In an aspect
of this embodiment, a Cathepsin L cleavage site comprises the
consensus sequence SEQ ID NO: 416, where X.sub.1 is W, an acidic
amino acid like D and E, an amidic amino acid like N and Q, a
positive amino acid like H, K, and R, an uncharged amino acid like
C, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.2 is any amino acid; X.sub.3 is L, V, F or Y;
and X.sub.4, X.sub.5, X.sub.6, X.sub.7, and X.sub.8 are any amino
acid. In another aspect of this embodiment, a Cathepsin L cleavage
site comprises the consensus sequence SEQ ID NO: 416, where X.sub.1
is G, P, A, L, Q, E, or K; X.sub.2 is an aromatic amino acid like
F, W, and Y, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; X.sub.3 is L, V, F or Y; X.sub.4 is G, A, F, T, Q,
E, K, or R; X.sub.5 is G, A, S, an acidic amino acid like D and E,
an amidic amino acid like N and Q, or a positive amino acid like H,
K, and R; X.sub.6 is P, A, L, I, S, Q, an acidic amino acid like D
and E, or a positive amino acid like H, K, and R; X.sub.7 is a
positive amino acid like H, K, and R, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; and X.sub.8 is P, L, S,
T, an acidic amino acid like D and E, an amidic amino acid like N
and Q, or a basic amino acid like K, and R. In another aspect of
this embodiment, a Cathepsin L cleavage site comprises the
consensus sequence SEQ ID NO: 416, where X.sub.1 is G, A, Q, E, or
K; X.sub.2 is G, P, L, or F; X.sub.3 is L, V, F or Y; X.sub.4 is G,
A, F, T, Q, E, K, or R; X.sub.5 is A, S, Q, E, K, or R; X.sub.6 is
P, A, L, I, S, or E; X.sub.7 P, L, or R; and X.sub.8 is P, L, S, or
K. In other aspects of this embodiment, a Cathepsin L cleavage site
comprises, e.g., SEQ ID NO: 417, SEQ ID NO: 418, SEQ ID NO: 419,
SEQ ID NO: 420, SEQ ID NO: 421, SEQ ID NO: 422, SEQ ID NO: 423, SEQ
ID NO: 424, SEQ ID NO: 425, SEQ ID NO: 426, SEQ ID NO: 427, SEQ ID
NO: 428, SEQ ID NO: 429, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO:
432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO:
436, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO:
440, SEQ ID NO: 441, SEQ ID NO: 442, or SEQ ID NO: 443.
[0288] Aspects of the present specification disclose, in part, a
PAR1 cleavage site as an inactivation cleavage site. As used
herein, the term "PAR1 cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
PAR1 under conditions suitable for PAR1 protease activity. It is
envisioned that any amino acid sequence cleaved by PAR1 can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a PAR1 cleavage
site is X.sub.1X.sub.2X.sub.3X.sub.4(K/R)X.sub.5 (SEQ ID NO: 444),
where X.sub.1 is preferentially a small non-polar amino acid like
A, C G, S, and T; X.sub.2 is preferentially a large non-polar amino
acid like F, I, L, M, V, or an aromatic amino acid like F, H, W, or
Y; X.sub.3 is preferentially a large non-polar amino acid like F,
I, L, M, V, or an aromatic amino acid like F, H, W, or Y; X.sub.4
is preferentially an aliphatic hydrophobic amino acid like, G, P,
A, V, L, I, and M; and X.sub.5 is preferentially an amidic amino
acid like N and Q, or an aromatic hydrophobic amino acid like F, W,
or Y. Table 4 lists exemplary reference cleavage sites for PAR1
(SEQ ID NO: 445-452). Additional PAR1 cleavage sites are well known
in the art or can be defined by routine methods.
[0289] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a PAR1 cleavage site. In an aspect of this
embodiment, a PAR1 cleavage site comprises the consensus sequence
SEQ ID NO: 444, where X.sub.1 is a small non-polar amino acid like
A, C G, S, and T; X.sub.2 is a large non-polar amino acid like F,
I, L, M, V, or an aromatic amino acid like F, H, W, or Y; X.sub.3
is a large non-polar amino acid like F, I, L, M, V, or an aromatic
amino acid like F, H, W, or Y; X.sub.4 is an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M; and X.sub.5 is an amidic
amino acid like N and Q, or an aromatic hydrophobic amino acid like
F, W, or Y. In another aspect of this embodiment, a PAR1 cleavage
site comprises the consensus sequence SEQ ID NO: 444, where X.sub.1
is S, T, or G; X.sub.2 is F or Y; X.sub.3 is L, P, or F; X.sub.4 is
A, G, I, or L; and X.sub.5 is F or N. In other aspects of this
embodiment, a PAR1 cleavage site comprises, e.g., SEQ ID NO: 445,
SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 449, SEQ
ID NO: 450, SEQ ID NO: 451, or SEQ ID NO: 452.
[0290] Aspects of the present specification disclose, in part, a
PAR2 cleavage site as an inactivation cleavage site. As used
herein, the term "PAR2 cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
PAR2 under conditions suitable for PAR2 protease activity. It is
envisioned that any amino acid sequence cleaved by PAR2 can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a PAR2 cleavage
site is X.sub.1X.sub.2X.sub.3X.sub.4(K/R)X.sub.5 (SEQ ID NO: 453),
where X.sub.1 is preferentially a small non-polar amino acid like
A, C G, S, and T; X.sub.2 is preferentially a large non-polar amino
acid like F, I, L, M, V; X.sub.3 is preferentially a large
non-polar amino acid like F, I, L, M, V; X.sub.4 is preferentially
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;
and X.sub.5 is preferentially a large non-polar amino acid like F,
I, L, M, V. Table 4 lists exemplary reference cleavage sites for
PAR2 (SEQ ID NO: 454-455). Additional PAR2 cleavage sites are well
known in the art or can be defined by routine methods.
[0291] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a PAR2 cleavage site. In an aspect of this
embodiment, a PAR2 cleavage site comprises the consensus sequence
SEQ ID NO: 453, where X.sub.1 is a small non-polar amino acid like
A, C G, S, and T; X.sub.2 is a large non-polar amino acid like F,
I, L, M, V; X.sub.3 is a large non-polar amino acid like F, I, L,
M, V; X.sub.4 is an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and M; and X.sub.5 is a large non-polar amino acid like F,
I, L, M, V. In another aspect of this embodiment, a PAR2 cleavage
site comprises the consensus sequence SEQ ID NO: 453, where X.sub.1
is S; X.sub.2 is I or L; X.sub.3 is I or L; X.sub.4 is A or G;
X.sub.5 is L or V. In other aspects of this embodiment, a PAR2
cleavage site comprises, e.g., SEQ ID NO: 454 or SEQ ID NO:
455.
[0292] Aspects of the present specification disclose, in part, a
PAR3 cleavage site as an inactivation cleavage site. As used
herein, the term "PAR3 cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
PAR3 under conditions suitable for PAR3 protease activity. It is
envisioned that any amino acid sequence cleaved by PAR3 can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a PAR3 cleavage
site is X.sub.1X.sub.2X.sub.3X.sub.4X.sub.6X.sub.6 (SEQ ID NO:
456), where X.sub.1 is preferentially a small non-polar amino acid
like A, C G, S, and T; X.sub.2 is preferentially a large non-polar
amino acid like F, I, L, M, V; X.sub.3 is preferentially an amidic
amino acid like N and Q, or a basic amino acid like K and R;
X.sub.4 is preferentially a small non-polar amino acid like A, C G,
S, and T; X.sub.5 is preferentially a small non-polar amino acid
like A, C G, S, and T, or a small polar amino acid like D, N, or P;
and X.sub.6 is preferentially an acidic amino acid like D and E, or
a small polar amino acid like D, N, or P. Table 4 lists exemplary
reference cleavage sites for PAR3 (SEQ ID NO: 457-459). Additional
PAR3 cleavage sites are well known in the art or can be defined by
routine methods.
[0293] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a PAR3 cleavage site. In an aspect of this
embodiment, a PAR3 cleavage site comprises the consensus sequence
SEQ ID NO: 456, where X.sub.1 is a small non-polar amino acid like
A, C G, S, and T; X.sub.2 is a large non-polar amino acid like F,
I, L, M, V; X.sub.3 is an amidic amino acid like N and Q, or a
basic amino acid like K and R; X.sub.4 is a small non-polar amino
acid like A, C G, S, and T; X.sub.5 is a small non-polar amino acid
like A, C G, S, and T, or a small polar amino acid like D, N, or P;
and X.sub.6 is an acidic amino acid like D and E, or a small polar
amino acid like D, N, or P. In another aspect of this embodiment, a
PAR3 cleavage site comprises the consensus sequence SEQ ID NO: 456,
where X.sub.1 is S or T; X.sub.2 is F; X.sub.3 is N or R; X.sub.4
is A or G; X.sub.5 is A, G, or N and X.sub.6 is P or E. In other
aspects of this embodiment, a PAR3 cleavage site comprises, e.g.,
SEQ ID NO: 457, SEQ ID NO: 458, or SEQ ID NO: 459.
[0294] Aspects of the present specification disclose, in part, a
PAR4 cleavage site as an inactivation cleavage site. As used
herein, the term "PAR4 cleavage site" refers to a scissile bond
together with adjacent or non-adjacent recognition elements, or
both, sufficient for detectable proteolysis at the scissile bond by
PAR4 under conditions suitable for PAR4 protease activity. It is
envisioned that any amino acid sequence cleaved by PAR4 can be
useful in aspects of the present specification. Although exceptions
are known, a generalized consensus sequence for a PAR4 cleavage
site is X.sub.1X.sub.2X.sub.3X.sub.4(K/R/Q/F)X.sub.5 (SEQ ID NO:
460), where X.sub.1 is preferentially a small non-polar amino acid
like A, C G, S, and T; X.sub.2 is preferentially a large non-polar
amino acid like F, I, L, M, V, or an aromatic amino acid like F, H,
W, or Y; X.sub.3 is preferentially an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and M; X.sub.4 is preferentially an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; and
X.sub.5 is preferentially a basic amino acid like K and R, an
aromatic hydrophobic amino acid like F, W, or Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. Table 4 lists
exemplary reference cleavage sites for PAR4 (SEQ ID NO: 461-478).
Additional PAR4 cleavage sites are well known in the art or can be
defined by routine methods.
[0295] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises a PAR4 cleavage site. In an aspect of this
embodiment, a PAR4 cleavage site comprises the consensus sequence
SEQ ID NO: 460, where X.sub.1 is a small non-polar amino acid like
A, C G, S, and T; X.sub.2 is a large non-polar amino acid like F,
I, L, M, V, or an aromatic amino acid like F, H, W, or Y; X.sub.3
is an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
M; X.sub.4 is an aliphatic hydrophobic amino acid like, G, P, A, V,
L, I, and M; and X.sub.5 is a basic amino acid like K and R, an
aromatic hydrophobic amino acid like F, W, or Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and M. In another
aspect of this embodiment, a PAR4 cleavage site comprises the
consensus sequence SEQ ID NO: 460, where X.sub.1 is A, G, S, or T;
X.sub.2 is F or Y; X.sub.3 is A or P; X.sub.4 is A or G; and
X.sub.5 is A, V, P, F, W, Y, or K. In other aspects of this
embodiment, a PAR4 cleavage site comprises, e.g., SEQ ID NO: 461,
SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ ID NO: 465, SEQ
ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID
NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO:
474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, or SEQ ID NO:
478.
[0296] The location of an inactivation cleavage site is a critical
aspect that is governed by several criteria. First, the placement
of the inactivation cleavage site should not substantially affect
the ability of a Clostridial toxin or Clostridial toxin chimeric to
intoxicate its target cell. As used herein, the term "not
substantially affect," with regards to intoxication, refers to a
Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification that can still execute the overall
intoxication mechanism whereby a Clostridial toxin or Clostridial
toxin chimeric enters a target cell and proteolytically cleaves a
target substrate and encompasses the binding of a Clostridial toxin
or Clostridial toxin chimeric to a low or high affinity receptor
complex, the internalization of the toxin/receptor complex, the
translocation of the light chain into the cytoplasm and the
enzymatic modification of a target substrate.
[0297] In an aspect of this embodiment, a Clostridial toxin or
Clostridial toxin chimeric comprising an inactivation cleavage site
can intoxicate a target cell to the same extent as the same or
similar Clostridial toxin or Clostridial toxin chimeric, but
without the inactivation cleavage site modification. In other
aspects of this embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprising an inactivation cleavage site can
intoxicate a target cell by, e.g., at least 50%, 60%, 70%, 80%, 90%
or 95% the extent as the same or similar Clostridial toxin or
Clostridial toxin chimeric, but without the inactivation cleavage
site modification. In other aspects of this embodiment, a
Clostridial toxin or Clostridial toxin chimeric comprising an
inactivation cleavage site can intoxicate a target cell by, e.g.,
at most 50%, 60%, 70%, 80%, 90% or 95% the extent as the same or
similar Clostridial toxin or Clostridial toxin chimeric, but
without the inactivation cleavage site modification.
[0298] Second, the placement of an inactivation cleavage site
should be at a surface exposed region of the toxin or Clostridial
toxin chimeric and not buried internally within the protein or
masked by secondary structure elements. Proper surface exposure of
the inactivation cleavage site facilitates proper access of the
site to its corresponding protease, thereby enabling proteolytic
cleavage. Proteolytic cleavage of the inactivation cleavage site by
its corresponding protease substantially inactivates the ability of
the Clostridial toxin or Clostridial toxin chimeric to intoxicate
the cell. As used herein, the term "substantially inactivates,"
with regards to intoxication, refers to a Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification
that, after cleavage at an inactivation cleavage site, has a
reduced ability to execute the overall intoxication mechanism
whereby a Clostridial toxin or Clostridial toxin chimeric enters a
target cell and proteolytically cleaves a target substrate and
encompasses the binding of a Clostridial toxin or Clostridial toxin
chimeric to a low or high affinity receptor complex, the
internalization of the toxin/receptor complex, the translocation of
the light chain into the cytoplasm and the enzymatic modification
of a target substrate.
[0299] In one aspect of this embodiment, proteolytic cleavage of a
Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification at an inactivation cleavage site results in
complete inability of the toxin to intoxicate a target cell as
compared to the same or similar Clostridial toxin or Clostridial
toxin chimeric, but in a proteolytic uncleaved state (La, the
intoxication cleavage site is intact or uncleaved). In other
aspects of this embodiment, proteolytic cleavage of a Clostridial
toxin or Clostridial toxin chimeric disclosed in the present
specification at an inactivation cleavage site results in, e.g., at
least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%
decreased ability to intoxicate a target cell as compared to the
same or similar Clostridial toxin or Clostridial toxin chimeric,
but in a proteolytic uncleaved state. In other aspects of this
embodiment, proteolytic cleavage of a Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification
at an inactivation cleavage site results in, e.g., at most a 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% decreased ability to
intoxicate a target cell as compared to the same or similar
Clostridial toxin or Clostridial toxin chimeric, but in a
proteolytic uncleaved state.
[0300] In an aspect of the present specification, an inactivation
cleavage site is located within an inactivation cleavage site
region. As used herein, the term "inactivation cleavage site
region" refers to an amino acid sequence of a Clostridial toxin or
Clostridial toxin chimeric that can be modified to contain an
inactivation cleavage site because such modification will not
substantially disrupt the ability of the protein to intoxicate a
target cell; and upon exposure to its cognate protease, the
inactivation cleavage site will be cleaved and substantially
inactivate the Clostridial toxin or Clostridial toxin chimeric. The
location of an inactivation cleavage site can be anywhere within
the inactivation cleavage site region, with the proviso that such
location will not substantially affect the ability of the
Clostridial toxin or Clostridial toxin chimeric to intoxicate a
target cell; and upon exposure to its cognate protease, cleavage of
the inactivation cleavage site will substantially inactivate the
Clostridial toxin or Clostridial toxin chimeric. Table 5 lists
exemplary inactivation cleavage site regions suitable for use with
a Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification.
[0301] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification comprises an
inactivation cleavage site located within inactivation cleavage
site region. In aspects of this embodiment, a Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification
comprises an inactivation cleavage site located within inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain.
[0302] In other aspects of this embodiment, a Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification
comprises an inactivation cleavage site is located within an
inactivation cleavage site region comprising amino acids 462-496 of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino
acids 458-492 of SEQ ID NO: 3; amino acids 464-487 of SEQ ID NO: 6,
SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino
acids 463-496 of SEQ ID NO: 11 or SEQ ID NO: 12; amino acids
458-491 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids 434-467 of
SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino acids 453-486
of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; amino acids
458-491 of SEQ ID NO: 21; amino acids 475-508 of SEQ ID NO: 22;
amino acids 443-476 of SEQ ID NO: 23; or amino acids 434-467 of SEQ
ID NO: 24 or SEQ ID NO: 25.
[0303] In yet other aspects of this embodiment, a Clostridial toxin
or Clostridial toxin chimeric disclosed in the present
specification comprises an inactivation cleavage site is located
within an inactivation cleavage site region comprising amino acids
618-634 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
5; amino acids 614-630 of SEQ ID NO: 3; amino acids 605-621 of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
10; amino acids 613-629 of SEQ ID NO: 11 or SEQ ID NO: 12; amino
acids 609-625 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids
587-603 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino
acids 604-620 of SEQ ID NO: 18; amino acids 605-621 of SEQ ID NO:
19 or SEQ ID NO: 20; amino acids 610-626 of SEQ ID NO: 21; amino
acids 627-643 of SEQ ID NO: 22; amino acids 596-612 of SEQ ID NO:
23; or amino acids 587-603 of SEQ ID NO: 24 or SEQ ID NO: 25.
TABLE-US-00005 TABLE 5 Inactivation Cleavage Site Regions of
Clostridial Toxins SEQ ID Inactivation Cleavage Site Regions Toxin
NO: 1 2 3 4 5 6 7 8 BoNT/ 1 L462- T618- G638- L665- N752- N826-
T844- K871- A L496 I634 D651 N687 N765 D835 L863 A895 BoNT/ 2 L464-
A605- G625- L652- N739- N813- Y831- S858- B P487 V621 N638 N674
D752 A824 I850 G882 BONT/ 3 L463- I613- G633- L660- K747- H821-
S839- N866- C1 S496 I629 N646 E682 Q760 D830 K858 N890 BoNT/ 4
L458- I609- G629- L656- K743- H817- S835- N862- D S491 I625 N642
E678 Q756 D826 K854 N886 BoNT/ 5 L434- A587- G607- L634- N724-
H800- T818- K845- E D467 V603 N620 N659 D739 Q809 I837 D869 BoNT/ 6
L453- A605- G625- L652- N742- H818- T836- K863- F N486 V621 N638
N677 N757 N827 I855 G887 BoNT/ 7 L458- S610- G630- M657- N744-
N818- H836- S863- G S491 I626 N643 N679 D757 N827 I855 G887 TeNT 8
L475- S627- G647- L674- K761- N835- V854- V879- S508 V643 N660 Q696
E774 K844 V871 N903 BaNT 9 L443- A596- G616- L643- N733- N809-
T828- K855- N476 V612 N629 S668 N748 P819 I847 G879 BuNT 10 L434-
A587- G607- L634- N724- H800- T818- K845- D467 V603 N620 S659 D739
Q809 I837 D869
[0304] In still other aspects of this embodiment, a Clostridial
toxin or Clostridial toxin chimeric disclosed in the present
specification comprises an inactivation cleavage site is located
within an inactivation cleavage site region comprising amino acids
638-651 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
5; amino acids 634-647 of SEQ ID NO: 3; amino acids 625-638 of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
10; amino acids 633-646 of SEQ ID NO: 11 or SEQ ID NO: 12; amino
acids 629-642 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids
607-620 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino
acids 624-637 of SEQ ID NO: 18; amino acids 625-638 of SEQ ID NO:
19 or SEQ ID NO: 20; amino acids 630-643 of SEQ ID NO: 21; amino
acids 647-660 of SEQ ID NO: 22; amino acids 616-629 of SEQ ID NO:
23; or amino acids 607-620 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0305] In further aspects of this embodiment, a Clostridial toxin
or Clostridial toxin chimeric disclosed in the present
specification comprises an inactivation cleavage site is located
within an inactivation cleavage site region comprising amino acids
665-687 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
5; amino acids 661-683 of SEQ ID NO: 3; amino acids 652-674 of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
10; amino acids 660-682 of SEQ ID NO: 11 or SEQ ID NO: 12; amino
acids 656-678 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids
634-659 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino
acids 651-676 of SEQ ID NO: 18; amino acids 652-677 of SEQ ID NO:
19 or SEQ ID NO: 20; amino acids 657-679 of SEQ ID NO: 21; amino
acids 674-696 of SEQ ID NO: 22; amino acids 643-668 of SEQ ID NO:
23; or amino acids 634-659 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0306] In other aspects of this embodiment, a Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification
comprises an inactivation cleavage site is located within an
inactivation cleavage site region comprising amino acids 752-765 of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino
acids 748-761 of SEQ ID NO: 3; amino acids 739-752 of SEQ ID NO: 6,
SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino
acids 747-760 of SEQ ID NO: 11 or SEQ ID NO: 12; amino acids
743-756 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids 724-739 of
SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino acids 741-756
of SEQ ID NO: 18; amino acids 742-757 of SEQ ID NO: 19 or SEQ ID
NO: 20; amino acids 744-757 of SEQ ID NO: 21; amino acids 761-774
of SEQ ID NO: 22; amino acids 733-748 of SEQ ID NO: 23; or amino
acids 724-739 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0307] In yet other aspects of this embodiment, a Clostridial toxin
or Clostridial toxin chimeric disclosed in the present
specification comprises an inactivation cleavage site is located
within an inactivation cleavage site region comprising amino acids
826-835 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
5; amino acids 824-831 of SEQ ID NO: 3; amino acids 813-824 of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
10; amino acids 821-830 of SEQ ID NO: 11 or SEQ ID NO: 12; amino
acids 817-826 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids
800-809 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino
acids 817-826 of SEQ ID NO: 18; amino acids 818-827 of SEQ ID NO:
19 or SEQ ID NO: 20; amino acids 818-827 of SEQ ID NO: 21; amino
acids 835-844 of SEQ ID NO: 22; amino acids 809-819 of SEQ ID NO:
23; or amino acids 800-809 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0308] In still other aspects of this embodiment, a Clostridial
toxin or Clostridial toxin chimeric disclosed in the present
specification comprises an inactivation cleavage site is located
within an inactivation cleavage site region comprising amino acids
844-863 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
5; amino acids 840-859 of SEQ ID NO: 3; amino acids 831-850 of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
10; amino acids 839-858 of SEQ ID NO: 11 or SEQ ID NO: 12; amino
acids 835-854 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids
818-837 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino
acids 835-854 of SEQ ID NO: 18; amino acids 836-855 of SEQ ID NO:
19 or SEQ ID NO: 20; amino acids 836-855 of SEQ ID NO: 21; amino
acids 854-871 of SEQ ID NO: 22; amino acids 828-847 of SEQ ID NO:
23; or amino acids 818-837 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0309] In further aspects of this embodiment, a Clostridial toxin
or Clostridial toxin chimeric disclosed in the present
specification comprises an inactivation cleavage site is located
within an inactivation cleavage site region comprising amino acids
871-895 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
5; amino acids 867-891 of SEQ ID NO: 3; amino acids 858-882 of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
10; amino acids 866-890 of SEQ ID NO: 11 or SEQ ID NO: 12; amino
acids 862-886 of SEQ ID NO: 13 or SEQ ID NO: 14; amino acids
845-869 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; amino
acids 862-886 of SEQ ID NO: 18; amino acids 863-887 of SEQ ID NO:
19 or SEQ ID NO: 20; amino acids 863-887 of SEQ ID NO: 21; amino
acids 879-903 of SEQ ID NO: 22; amino acids 855-879 of SEQ ID NO:
23; or amino acids 845-869 of SEQ ID NO: 24 or SEQ ID NO: 25.
[0310] In another aspect of this embodiment, a BoNT/A or BoNT/A
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BoNT/A or BoNT/A
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 462-496, 618-634,
638-651, 665-687, 752-765, 826-835, 844-863, or 871-895 of SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; or comprising
amino acids 458-492, 614-630, 634-647, 665-687, 748-761, 822-831,
840-859, or 867-891 of SEQ ID NO: 3. In yet other aspects of this
embodiment, a BoNT/A comprising an inactivation cleavage site
located within inactivation cleavage site region is encoded by SEQ
ID NO: 530, SEQ ID NO: 532, SEQ ID NO: 534, or SEQ ID NO: 536. In
still other aspects of this embodiment, a BoNT/A comprising an
inactivation cleavage site located within inactivation cleavage
site region comprises SEQ ID NO: 531, SEQ ID NO: 533, SEQ ID NO:
535, or SEQ ID NO: 537.
[0311] In yet another aspect of this embodiment, a BoNT/B or BoNT/B
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BoNT/B or BoNT/B
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 464-487, 605-621,
625-638, 652-674, 739-752, 813-824, 831-850, or 858-882 of SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
10.
[0312] In still another aspect of this embodiment, a BoNT/C1 or
BoNT/C1 chimeric disclosed in the present specification comprises
an inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BoNT/C1 or
BoNT/C1 chimeric disclosed in the present specification comprises
an inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 463-496, 613-629,
633-646, 660-682, 747-760, 821-830, 839-858, or 866-890 of SEQ ID
NO: 11 or SEQ ID NO: 12.
[0313] In a further aspect of this embodiment, a BoNT/D or BoNT/D
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BoNT/D or BoNT/D
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 458-491, 609-625,
629-642, 656-678, 743-756, 817-826, 835-854, or 862-886 of SEQ ID
NO: 13 or SEQ ID NO: 14.
[0314] In another aspect of this embodiment, a BoNT/E or BoNT/E
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BoNT/E or BoNT/E
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 434-467, 587-603,
607-620, 634-659, 724-739, 800-809, 818-837, or 845-869 of SEQ ID
NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
[0315] In still another aspect of this embodiment, a BoNT/F or
BoNT/F chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BoNT/F or BoNT/F
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 453-486, 604-620,
624-637, 651-676, 741-756, 817-826, 835-854, or 862-886 of SEQ ID
NO: 18; or comprising amino acids 453-486, 605-621, 625-638,
652-677, 742-757, 818-827, 836-855, or 863-887 of SEQ ID NO: 19 or
SEQ ID NO: 20.
[0316] In a further aspect of this embodiment, a BoNT/G or BoNT/G
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BoNT/G or BoNT/G
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 458-491, 610-626,
630-643, 657-679, 744-757, 818-827, 836-855, or 863-887 of SEQ ID
NO: 21.
[0317] In another aspect of this embodiment, a TeNT or TeNT
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a TeNT or TeNT
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 475-508, 627-643,
647-660, 674-696, 761-774, 835-844, 854-871, or 879-903 of SEQ ID
NO: 22.
[0318] In yet another aspect of this embodiment, a BaNT or BaNT
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BaNT or BaNT
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 443-476, 596-612,
616-629, 643-668, 733-748, 809-819, 828-847, or 855-879 of SEQ ID
NO: 23.
[0319] In still another aspect of this embodiment, a BuNT or BuNT
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region from the translocation domain or the H.sub.CN
subdomain. In other aspects of this embodiment, a BuNT or BuNT
chimeric disclosed in the present specification comprises an
inactivation cleavage site is located within an inactivation
cleavage site region comprising amino acids 434-467, 587-603,
607-620, 634-659, 724-739, 800-809, 818-837, or 845-869 of SEQ ID
NO: 24 or SEQ ID NO: 25.
[0320] In an aspect of the present specification, a Clostridial
toxin or Clostridial toxin chimeric comprising an inactivation
cleavage site has a safety margin greater than the safety margin
for the same or similar Clostridial toxin or Clostridial toxin
chimeric, but without the inactivation cleavage site. In other
words, the addition of an inactivation cleavage site increases the
safety margin of the Clostridial toxin or Clostridial toxin
chimeric relative to the same or similar Clostridial toxin or
Clostridial toxin chimeric, but without the additional inactivation
cleavage site.
[0321] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprising an inactivation cleavage site has a
safety margin that is greater relative to the same or similar
Clostridial toxin or Clostridial toxin chimeric, but without the
inactivation cleavage site. In aspects of this embodiment, a
Clostridial toxin or Clostridial toxin chimeric comprising an
inactivation cleavage site has a safety margin that is greater
than, e.g., at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 100%, 110%, at least 120%, at least 130%, at least
140%, at least 150%, at least 160%, at least 170%, at least 180%,
at least 190%, at least 200%, 210%, at least 220%, at least 230%,
at least 240%, at least 250%, at least 260%, at least 270%, at
least 280%, at least 290%, or at least 300%, relative to the same
or similar Clostridial toxin or Clostridial toxin chimeric, but
without the inactivation cleavage site. In other aspects of this
embodiment, a Clostridial toxin or Clostridial toxin chimeric
comprising an inactivation cleavage site has a safety margin that
is greater than, e.g., at most 10%, at most 20%, at most 30%, at
most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at
most 90%, at most 100%, 110%, at most 120%, at most 130%, at most
140%, at most 150%, at most 160%, at most 170%, at most 180%, at
most 190%, at most 200%, 210%, at most 220%, at most 230%, at most
240%, at most 250%, at most 260%, at most 270%, at most 280%, at
most 290%, or at most 300%, relative to the same or similar
Clostridial toxin or Clostridial toxin chimeric, but without the
inactivation cleavage site. In yet other aspects of this
embodiment, a Clostridial toxin or Clostridial toxin chimeric
comprising an inactivation cleavage site has a safety margin that
is greater by, e.g., about 10% to about 300%, about 20% to about
300%, about 30% to about 300%, about 40% to about 300%, about 50%
to about 300%, about 60% to about 300%, about 70% to about 300%,
about 80% to about 300%, about 90% to about 300%, or about 100% to
about 300%, relative to the same or similar Clostridial toxin or
Clostridial toxin chimeric, but without the inactivation cleavage
site.
[0322] In other aspects embodiment, a Clostridial toxin or
Clostridial toxin chimeric comprising an inactivation cleavage site
has a safety margin that is greater than, e.g., at least 1-fold, at
least 1-fold, at least 3-fold, at least 4-fold, at least 5-fold, at
least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or
at least 10-fold, relative to the same or similar Clostridial toxin
or Clostridial toxin chimeric, but without the inactivation
cleavage site. In yet other aspects embodiment, a Clostridial toxin
or Clostridial toxin chimeric comprising an inactivation cleavage
site has a safety margin that is greater than, e.g., at least
1-fold, at most 1-fold, at most 3-fold, at most 4-fold, at most
5-fold, at most 6-fold, at most 7-fold, at most 8-fold, at most
9-fold, or at most 10-fold, relative to the same or similar
Clostridial toxin or Clostridial toxin chimeric, but without the
inactivation cleavage site. In still other aspects of this
embodiment, a Clostridial toxin or Clostridial toxin chimeric
comprising an inactivation cleavage site has a safety margin that
is greater by, e.g., about 1-fold to about 10-fold, about 1-fold to
about 9-fold, about 1-fold to about 8-fold, about 1-fold to about
7-fold, about 1-fold to about 6-fold, about 1-fold to about 5-fold,
about 2-fold to about 10-fold, about 2-fold to about 9-fold, about
2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold
to about 6-fold, or about 2-fold to about 5-fold.
[0323] In another embodiment, a Clostridial toxin or Clostridial
toxin chimeric comprises the addition of an inactivation cleavage
site that increases the safety margin of the Clostridial toxin or
Clostridial toxin chimeric relative to the same or similar
Clostridial toxin or Clostridial toxin chimeric, but without the
additional inactivation cleavage site. In aspects of this
embodiment, a Clostridial toxin or Clostridial toxin chimeric
comprises the addition of an inactivation cleavage site that
increases the safety margin of the Clostridial toxin or Clostridial
toxin chimeric relative to the same or similar Clostridial toxin or
Clostridial toxin chimeric, but without the additional inactivation
cleavage site by, e.g., at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 100%, 110%, at least 120%, at least
130%, at least 140%, at least 150%, at least 160%, at least 170%,
at least 180%, at least 190%, at least 200%, 210%, at least 220%,
at least 230%, at least 240%, at least 250%, at least 260%, at
least 270%, at least 280%, at least 290%, or at least 300%. In
other aspects of this embodiment, a Clostridial toxin or
Clostridial toxin chimeric comprises the addition of an
inactivation cleavage site that increases the safety margin of the
Clostridial toxin or Clostridial toxin chimeric relative to the
same or similar Clostridial toxin or Clostridial toxin chimeric,
but without the additional inactivation cleavage site by, e.g., at
most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at
most 60%, at most 70%, at most 80%, at most 90%, at most 100%,
110%, at most 120%, at most 130%, at most 140%, at most 150%, at
most 160%, at most 170%, at most 180%, at most 190%, at most 200%,
210%, at most 220%, at most 230%, at most 240%, at most 250%, at
most 260%, at most 270%, at most 280%, at most 290%, or at most
300%. In yet other aspects of this embodiment, a Clostridial toxin
or Clostridial toxin chimeric comprises the addition of an
inactivation cleavage site that increases the safety margin of the
Clostridial toxin or Clostridial toxin chimeric relative to the
same or similar Clostridial toxin or Clostridial toxin chimeric,
but without the additional inactivation cleavage site by, e.g.,
about 10% to about 300%, about 20% to about 300%, about 30% to
about 300%, about 40% to about 300%, about 50% to about 300%, about
60% to about 300%, about 70% to about 300%, about 80% to about
300%, about 90% to about 300%, or about 100% to about 300%.
[0324] In other aspects of this embodiment, a Clostridial toxin or
Clostridial toxin chimeric comprises the addition of an
inactivation cleavage site that increases the safety margin of the
Clostridial toxin or Clostridial toxin chimeric relative to the
same or similar Clostridial toxin or Clostridial toxin chimeric,
but without the additional inactivation cleavage site by, e.g., at
least 1-fold, at least 1-fold, at least 3-fold, at least 4-fold, at
least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at
least 9-fold, or at least 10-fold. In yet other aspects of this
embodiment, a Clostridial toxin or Clostridial toxin chimeric
comprises the addition of an inactivation cleavage site that
increases the safety margin of the Clostridial toxin or Clostridial
toxin chimeric relative to the same or similar Clostridial toxin or
Clostridial toxin chimeric, but without the additional inactivation
cleavage site by, e.g., at most 1-fold, at most 3-fold, at most
4-fold, at most 5-fold, at most 6-fold, at most 7-fold, at most
8-fold, at most 9-fold, or at most 10-fold. In still other aspects
of this embodiment, a Clostridial toxin or Clostridial toxin
chimeric comprises the addition of an inactivation cleavage site
that increases the safety margin of the Clostridial toxin or
Clostridial toxin chimeric relative to the same or similar
Clostridial toxin or Clostridial toxin chimeric, but without the
additional inactivation cleavage site by, e.g., about 1-fold to
about 10-fold, about 1-fold to about 9-fold, about 1-fold to about
8-fold, about 1-fold to about 7-fold, about 1-fold to about 6-fold,
about 1-fold to about 5-fold, about 2-fold to about 10-fold, about
2-fold to about 9-fold, about 2-fold to about 8-fold, about 2-fold
to about 7-fold, about 2-fold to about 6-fold, or about 2-fold to
about 5-fold.
[0325] In another embodiment, an inactivation cleavage site region
can be modified to include a single inactivation cleavage site. In
yet another embodiment, an inactivation cleavage site region can be
modified to include a plurality inactivation cleavage site cleavage
sites. In aspects of this embodiment, an inactivation cleavage site
cleavage site region can comprise, e.g., at least 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 inactivation cleavage sites. In other aspects of
this embodiment, an inactivation cleavage site cleavage site region
can comprise, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
inactivation cleavage sites. In yet other aspects of this
embodiment, an inactivation cleavage site cleavage site region can
comprise, e.g., 2-10 inactivation cleavage sites, 2-8 inactivation
cleavage sites, 2-6 inactivation cleavage sites, 2-4 inactivation
cleavage sites, 2-3 inactivation cleavage sites, 3-9 inactivation
cleavage sites, 3-7 inactivation cleavage sites, 3-5 inactivation
cleavage sites, or 3-4 inactivation cleavage sites.
[0326] In another embodiment, an inactivation cleavage site region
can be modified to include only one type of inactivation cleavage
site, such as, e.g., a thrombin cleavage site. In still another
embodiment, an inactivation cleavage site region can be modified to
include a plurality of different types of inactivation cleavage
sites, such as, e.g., a thrombin cleavage site, a Factor Xa
cleavage site, MMP-2 cleavage site, and a MMP-9 cleavage site. In
aspects of this embodiment, an inactivation cleavage site region
can comprise, e.g., at least 2, 3, 4, or 5 different types of
inactivation cleavage sites. In other aspects of this embodiment,
an inactivation cleavage site region can comprise, e.g., at most 2,
3, 4, or 5 different types of inactivation cleavage sites. In other
aspects of this embodiment, an inactivation cleavage site region
can comprise, e.g., 2-5 different types of inactivation cleavage
sites, 2-4 different types of inactivation cleavage sites, 2-3
different types of inactivation cleavage sites, 3-5 different types
of inactivation cleavage sites, or 3-4 different types of
inactivation cleavage sites.
[0327] Modification of an inactivation cleavage site region to
include a inactivation cleavage site can be accomplished by
altering at least one of the amino acids within the inactivation
cleavage site region. Non-limiting examples of an amino acid
alteration include a deletion of an amino acid, an addition of an
amino acid, or a substitution of an original amino acid with a
different amino acid. In aspects of this embodiment, an
inactivation cleavage site region is modified to include an
inactivation cleavage site by altering, e.g., at least 1, 2, 3, 4,
or 5 amino acids within the inactivation cleavage site region. In
other aspects of this embodiment, an inactivation cleavage site
region is modified to include an inactivation cleavage site by
altering, e.g., at most 1, 2, 3, 4, or 5 amino acids within the
inactivation cleavage site region. In yet aspects of this
embodiment, an inactivation cleavage site region is modified to
include an inactivation cleavage site by altering, e.g., 1-5 amino
acids within the inactivation cleavage site region, 1-4 amino acids
within the inactivation cleavage site region, 1-3 amino acids
within the inactivation cleavage site region, 1-2 amino acids
within the inactivation cleavage site region, 2-5 amino acids
within the inactivation cleavage site region, 2-4 amino acids
within the inactivation cleavage site region, 2-3 amino acids
within the inactivation cleavage site region, 3-5 amino acids
within the inactivation cleavage site region, or 4-5 amino acids
within the inactivation cleavage site region.
[0328] In aspects of this embodiment, an inactivation cleavage site
region is modified to include an inactivation cleavage site by
deleting, adding, substituting, or any combination thereof, e.g.,
at least 1, 2, 3, 4, or 5 amino acids within the inactivation
cleavage site region. In other aspects of this embodiment, an
inactivation cleavage site region is modified to include an
inactivation cleavage site by deleting, adding, substituting, or
any combination thereof, e.g., at most 1, 2, 3, 4, or 5 amino acids
within the inactivation cleavage site region. In yet aspects of
this embodiment, an inactivation cleavage site region is modified
to include an inactivation cleavage site by deleting, adding,
substituting, or any combination thereof, e.g., 1-5 amino acids
within the inactivation cleavage site region, 1-4 amino acids
within the inactivation cleavage site region, 1-3 amino acids
within the inactivation cleavage site region, 1-2 amino acids
within the inactivation cleavage site region, 2-5 amino acids
within the inactivation cleavage site region, 2-4 amino acids
within the inactivation cleavage site region, 2-3 amino acids
within the inactivation cleavage site region, 3-5 amino acids
within the inactivation cleavage site region, or 4-5 amino acids
within the inactivation cleavage site region.
[0329] Modification of an inactivation cleavage site region to
include an inactivation cleavage site can be achieved using
standard mutagenesis procedures known to a person skilled in the
art. Non-limiting examples of mutagenesis procedures, as well as
well-characterized reagents, conditions and protocols are readily
available from commercial vendors that include, without limitation,
BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences
Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.;
QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif.
These protocols are routine procedures within the scope of one
skilled in the art and from the teaching herein.
[0330] As mentioned above, Clostridial toxins and Clostridial toxin
chimeras disclosed in the present specification are translated as
single-chain polypeptides that are subsequently cleaved by
proteolytic scission within a disulfide loop region. This
posttranslational processing yields a di-chain molecule held
together by a single disulphide bond and noncovalent interactions.
The proteolytic scission within a disulfide loop region can be
achieved by using the endogenous protease cleavage sites
naturally-occurring within the di-chain loop region, or by
engineering the di-chain loop region to comprise an exogenous
protease cleavage site.
[0331] Aspects of the present specification disclose, in part, a
di-chain loop region. As used herein, the term "di-chain loop
region" refers to an amino acid sequence of a Clostridial toxin or
Clostridial toxin chimeric flanked by cysteine amino acids and
containing a protease cleavage site used to convert the
single-chain form of a Clostridial toxin or Clostridial toxin
chimeric into its di-chain form (Table 6). Non-limiting examples of
a di-chain loop region, include, a di-chain loop region of BoNT/A
comprising amino acids 430-454 of SEQ ID NO: 1; a di-chain loop
region of BoNT/B comprising amino acids 437-446 of SEQ ID NO: 2; a
di-chain loop region of BoNT/C1 comprising amino acids 437-453 of
SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino
acids 437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E
comprising amino acids 412-426 of SEQ ID NO: 5; a di-chain loop
region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; a
di-chain loop region of BoNT/G comprising amino acids 436-450 of
SEQ ID NO: 7; and a di-chain loop region of TeNT comprising amino
acids 439-467 of SEQ ID NO: 8 (Table 6).
TABLE-US-00006 TABLE 6 Di-chain Loop Region Di-chain Loop Region
Containing the Naturally-occurring Protease Toxin Cleavage Site
BoNT/A CVRGIITSKTKSLDKGYNK*----ALNDLC BoNT/B
CKSVK*-------------------APGIC BoNT/C1
CHKAIDGRSLYNK*------------TLDC BoNT/D
CLRLTKNSR*---------------DDSTC BoNT/E
CKNIVSVKGIR*--------------KSIC BoNT/F
CKSVIPRKGTK*------------APPRLC BoNT/G
CKPVMYKNTGK*--------------SEQC TeNT CKKIIPPTNIRENLYNRTA*SLIDLGGELC
BaNT CKS-IVSKKGTK*-------------NSLC BuNT
CKN-IVSVKGIR*-------------KSIC The amino acid sequence displayed
are as follows: BoNT/A, residues 430-454 of SEQ ID NO: 1; BoNT/B,
residues 437-446 of SEQ ID NO: 2; BoNT/C1, residues 437-453 of SEQ
ID NO: 3; BoNT/D, residues 437-450 of SEQ ID NO: 4; BoNT/E,
residues 412-426 of SEQ ID NO: 5; BoNT/F, residues 429-445 of SEQ
ID NO: 6; BoNT/G, residues 436-450 of SEQ ID NO: 7; TeNT, residues
439-467 of SEQ ID NO: 8; BaNT, residues 421-435 of SEQ ID NO: 9;
and BuNT, residues 412-426 of SEQ ID NO: 10. An asterisks (*)
indicates the peptide bond that is cleaved by a Clostridial toxin
protease.
[0332] Thus, in an embodiment, a di-chain loop region comprises a
Clostridial toxin di-chain loop region. In aspects of this
embodiment, a di-chain loop region comprises, e.g., a BoNT/A
di-chain loop region, a BoNT/B di-chain loop region, a BoNT/C1
di-chain loop region, a BoNT/D di-chain loop region, a BoNT/E
di-chain loop region, a BoNT/F di-chain loop region, a BoNT/G
di-chain loop region, a TeNT di-chain loop region, a BaNT di-chain
loop region, or a BuNT di-chain loop region. In other aspects of
this embodiment, a di-chain loop region comprises, e.g., a BoNT/A
di-chain loop region comprising amino acids 430-454 of SEQ ID NO:
1; a BoNT/B di-chain loop region comprising amino acids 437-446 of
SEQ ID NO: 2; a BoNT/C1 di-chain loop region comprising amino acids
437-453 of SEQ ID NO: 3; a BoNT/D di-chain loop region comprising
amino acids 437-450 of SEQ ID NO: 4; a BoNT/E di-chain loop region
comprising amino acids 412-426 of SEQ ID NO: 5; a BoNT/F di-chain
loop region comprising amino acids 429-445 of SEQ ID NO: 6; a
BoNT/G di-chain loop region comprising amino acids 436-450 of SEQ
ID NO: 7; or a TeNT di-chain loop region comprising amino acids
439-467 of SEQ ID NO: 8. a BaNT di-chain loop region comprising
amino acids 421-435 of SEQ ID NO: 9; or a BuNT di-chain loop region
comprising amino acids 412-426 of SEQ ID NO: 10.
[0333] Aspects of the present specification disclose, in part, an
endogenous di-chain loop protease cleavage site. As used herein,
the term "endogenous di-chain loop protease cleavage site" is
synonymous with a "naturally occurring di-chain loop protease
cleavage site" and refers to a naturally occurring protease
cleavage site found within the di-chain loop region of a naturally
occurring Clostridial toxin or Clostridial toxin chimeric and
includes, without limitation, naturally occurring Clostridial toxin
di-chain loop protease cleavage site variants, such as, e.g.,
Clostridial toxin di-chain loop protease cleavage site isoforms and
Clostridial toxin di-chain loop protease cleavage site subtypes.
Non-limiting examples of an endogenous protease cleavage site,
include, e.g., a BoNT/A di-chain loop protease cleavage site, a
BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain
loop protease cleavage site, a BoNT/D di-chain loop protease
cleavage site, a BoNT/E di-chain loop protease cleavage site, a
BoNT/F di-chain loop protease cleavage site, a BoNT/G di-chain loop
protease cleavage site and a TeNT di-chain loop protease cleavage
site.
[0334] While the identity of the protease is currently unknown, the
di-chain loop protease cleavage site for many Clostridial toxins
has been determined. In BoNTs, cleavage at K448-A449 converts the
single polypeptide form of BoNT/A into the di-chain form; cleavage
at K441-A442 converts the single polypeptide form of BoNT/B into
the di-chain form; cleavage at K449-T450 converts the single
polypeptide form of BoNT/C1 into the di-chain form; cleavage at
R445-D446 converts the single polypeptide form of BoNT/D into the
di-chain form; cleavage at R422-K423 converts the single
polypeptide form of BoNT/E into the di-chain form; cleavage at
K439-A440 converts the single polypeptide form of BoNT/F into the
di-chain form; and cleavage at K446-5447 converts the single
polypeptide form of BoNT/G into the di-chain form. Proteolytic
cleavage of the single polypeptide form of TeNT at A457-S458
results in the di-chain form. Proteolytic cleavage of the single
polypeptide form of BaNT at K431-N432 results in the di-chain form.
Proteolytic cleavage of the single polypeptide form of BuNT at
R422-K423 results in the di-chain form. Such a di-chain loop
protease cleavage site is operably-linked to a Clostridial toxin or
Clostridial toxin chimeric as a fusion protein. However, it should
also be noted that additional cleavage sites within the di-chain
loop also appear to be cleaved resulting in the generation of a
small peptide fragment being lost. As a non-limiting example,
BoNT/A single-chain polypeptide cleave ultimately results in the
loss of a ten amino acid fragment within the di-chain loop.
[0335] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification comprises a
di-chain loop region including an endogenous di-chain loop protease
cleavage site. In aspects of this embodiment, an endogenous
di-chain loop protease cleavage site located within the di-chain
loop region comprises, e.g., a BoNT/A di-chain loop protease
cleavage site, a BoNT/B di-chain loop protease cleavage site, a
BoNT/C1 di-chain loop protease cleavage site, a BoNT/D di-chain
loop protease cleavage site, a BoNT/E di-chain loop protease
cleavage site, a BoNT/F di-chain loop protease cleavage site, a
BoNT/G di-chain loop protease cleavage site, a TeNT di-chain loop
protease cleavage site, a BaNT di-chain loop protease cleavage
site, or a BuNT di-chain loop protease cleavage site. In other
aspects of this embodiment, an endogenous di-chain loop protease
cleavage site located within the di-chain loop region comprises,
e.g., a di-chain loop region of BoNT/A comprising amino acids
430-454 of SEQ ID NO: 1; a di-chain loop region of BoNT/B
comprising amino acids 437-446 of SEQ ID NO: 2; a di-chain loop
region of BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a
di-chain loop region of BoNT/D comprising amino acids 437-450 of
SEQ ID NO: 4; a di-chain loop region of BoNT/E comprising amino
acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/F
comprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop
region of BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; or
a di-chain loop region of TeNT comprising amino acids 439-467 of
SEQ ID NO: 8. a di-chain loop region of BaNT comprising amino acids
421-435 of SEQ ID NO: 9; or a di-chain loop region of BuNT
comprising amino acids 412-426 of SEQ ID NO: 10.
[0336] Aspects of the present specification disclose, in part, an
exogenous protease cleavage site. As used herein, the term
"exogenous protease cleavage site" is synonymous with "engineered
protease cleavage site", "non-naturally occurring protease cleavage
site", or "non-native protease cleavage site" and refers to a
protease cleavage site that is not normally present in a di-chain
loop region from a naturally occurring Clostridial toxin. Such
engineered or exogenous protease cleavage sites within the di-chain
loop region are used to convert the single-chain polypeptide form
of a Clostridial toxin of Clostridial toxin chimeric disclosed in
the present specification into its di-chain form. It is envisioned
that any and all exogenous protease cleavage sites can be used to
convert the single-chain polypeptide form of a Clostridial toxin or
Clostridial toxin chimeric into its active di-chain form are useful
to practice aspects of the present specification. Non-limiting
examples of exogenous protease cleavage sites include, e.g., a
plant papain cleavage site, an insect papain cleavage site, a
crustacean papain cleavage site, an enterokinase cleavage site, a
human rhinovirus 3C protease cleavage site, a human enterovirus 3C
protease cleavage site, a tobacco etch virus (TEV) protease
cleavage site, a Tobacco Vein Mottling Virus (TVMV) cleavage site,
a subtilisin cleavage site, a hydroxylamine cleavage site, or a
Caspase 3 cleavage site. Engineered protease cleavage sites located
within the di-chain loop are described in, e.g., Dolly, et al.,
Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,419,676,
Dolly, et al., Activatable Recombinant Neurotoxins, U.S. Pat. No.
7,422,877, Steward, et al., Activatable Recombinant Neurotoxins,
U.S. Patent Publication 2009/0069238, Steward, et al., Activatable
Recombinant Neurotoxins, U.S. Patent Publication 2008/0032930,
Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent
Publication 2009/0018081, Steward, et al., Activatable Recombinant
Neurotoxins, U.S. Patent Publication 2009/0005313, Steward, et al.,
Activatable Recombinant Neurotoxins, U.S. Patent Publication
2009/0004224; each of which is hereby incorporated by reference in
its entirety.
[0337] It is envisioned that an exogenous protease cleavage site of
any and all lengths can be useful in aspects of the present
specification with the proviso that the exogenous protease cleavage
site can be cleaved by its respective protease. Thus, in aspects of
this embodiment, an exogenous protease cleavage site can have a
length of, e.g., at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,
or at least 60 amino acids; or at most 6, 7, 8, 9, 10, 15, 20, 25,
30, 40, 50, or at least 60 amino acids.
[0338] In an embodiment, a Clostridial toxin or Clostridial toxin
chimeric disclosed in the present specification comprises a
di-chain loop region including an exogenous protease cleavage site.
In aspects of this embodiment, an exogenous protease cleavage site
located within the di-chain loop region comprises, e.g., a plant
papain cleavage site, an insect papain cleavage site, a crustacean
papain cleavage site, a non-human enterokinase protease cleavage
site, a Tobacco Etch Virus protease cleavage site, a Tobacco Vein
Mottling Virus protease cleavage site, a human rhinovirus 3C
protease cleavage site, a human enterovirus 3C protease cleavage
site, a subtilisin cleavage site, a hydroxylamine cleavage site, a
SUMO/ULP-1 protease cleavage site, and a non-human Caspase 3
cleavage site.
[0339] In an aspect of this embodiment, an exogenous protease
cleavage site located within the di-chain loop region comprises,
e.g., a non-human enterokinase cleavage site. In another aspect of
the embodiment, an exogenous protease cleavage site located within
the di-chain loop region comprises, e.g., a bovine enterokinase
protease cleavage site. In yet another aspect of the embodiment, an
exogenous protease cleavage site located within the di-chain loop
region comprises, e.g., SEQ ID NO: 480.
[0340] In another aspect of this embodiment, an exogenous protease
cleavage site located within the di-chain loop region comprises,
e.g., a Tobacco Etch Virus protease cleavage. In other aspects of
the embodiment, an exogenous protease cleavage site located within
the di-chain loop region comprises, e.g., the consensus sequence
EX.sub.1X.sub.2YX.sub.3Q*G (SEQ ID NO: 481) or
EX.sub.1X.sub.2YX.sub.3Q*S (SEQ ID NO: 482), where X.sub.1, X.sub.2
and X.sub.3 is any amino acid. In other aspects of the embodiment,
an exogenous protease cleavage site located within the di-chain
loop region comprises, e.g., SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID
NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 488, SEQ ID NO:
489, SEQ ID NO: 490, SEQ ID NO: 491, or SEQ ID NO: 492.
[0341] In another aspect of this embodiment, an exogenous protease
cleavage site located within the di-chain loop region comprises,
e.g., a Tobacco Vein Mottling Virus protease cleavage site. In
other aspects of the embodiment, an exogenous protease cleavage
site located within the di-chain loop region comprises, e.g., the
consensus sequence X.sub.1X.sub.2VRFQ*G (SEQ ID NO: 493) or
X.sub.1X.sub.2VRFQ*S (SEQ ID NO: 494), where X.sub.1 and X.sub.2
are independently any amino acid. In other aspects of the
embodiment, an exogenous protease cleavage site located within the
di-chain loop region comprises, e.g., SEQ ID NO: 495, SEQ ID NO:
496, SEQ ID NO: 497, or SEQ ID NO: 498.
[0342] In still another aspect of this embodiment, an exogenous
protease cleavage site located within the di-chain loop region
comprises, e.g., a human rhinovirus 3C protease cleavage site. In
another aspect of the embodiment, an exogenous protease cleavage
site located within the di-chain loop region comprises, e.g., the
consensus sequence X.sub.1X.sub.2LFQ*GP (SEQ ID NO: 499), where
X.sub.1 is any amino acid with an acidic amino acid like D or E
preferred; and X.sub.2 is preferentially S, T, and an aliphatic
hydrophobic amino acid like G, P, A, V, L, I, and M. In other
aspects of the embodiment, an exogenous protease cleavage site
located within the di-chain loop region comprises, e.g., SEQ ID NO:
500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO: 503, SEQ ID NO:
504, or SEQ ID NO: 505. In another aspect of the embodiment, an
exogenous protease cleavage site located within the di-chain loop
region comprises, e.g., a human rhinovirus 3C protease cleaved by
PRESCISSION.RTM..
[0343] In yet another aspect of this embodiment, an exogenous
protease cleavage site located within the di-chain loop region
comprises, e.g., a subtilisin cleavage site. In other aspects of
the embodiment, an exogenous protease cleavage site located within
the di-chain loop region comprises, e.g., the consensus sequence
X.sub.1X.sub.2X.sub.3X.sub.4H*Y (SEQ ID NO: 506) or
X.sub.1X.sub.2X.sub.3X.sub.4YH* (SEQ ID NO: 507), where X.sub.1,
X.sub.2, X.sub.3, and X.sub.4 are independently any amino acid. In
other aspects of the embodiment, an exogenous protease cleavage
site located within the di-chain loop region comprises, e.g., SEQ
ID NO: 508, SEQ ID NO: 509, or SEQ ID NO: 510. In other aspects of
the embodiment, an exogenous protease cleavage site located within
the di-chain loop region comprises, e.g., a subtilisin cleavage
site cleaved by GENENASE.RTM..
[0344] In yet another aspect of this embodiment, an exogenous
protease cleavage site located within the di-chain loop region
comprises, e.g., a hydroxylamine cleavage site. In other aspects of
the embodiment, an exogenous protease cleavage site located within
the di-chain loop region comprises, e.g., the dipeptide N*G. In
other aspects of the embodiment, an exogenous protease cleavage
site located within the di-chain loop region comprises, e.g., SEQ
ID NO: 511 or SEQ ID NO: 512.
[0345] In yet another aspect of this embodiment, an exogenous
protease cleavage site located within the di-chain loop region
comprises, e.g., a SUMO/ULP-1 protease cleavage site. In other
aspects of the embodiment, an exogenous protease cleavage site
located within the di-chain loop region comprises, e.g., the
consensus sequence GG*X.sub.1X.sub.2X.sub.3 (SEQ ID NO: 513), where
X.sub.1, X.sub.2, and X.sub.3 are independently any amino acid. In
other aspects of the embodiment, an exogenous protease cleavage
site located within the di-chain loop region comprises, e.g., SEQ
ID NO: 514.
[0346] In an aspect of this embodiment, an exogenous protease
cleavage site located within the di-chain loop region comprises,
e.g., a Caspase 3 cleavage site. In other aspects of the
embodiment, an exogenous protease cleavage site located within the
di-chain loop region comprises, e.g., a non-human Caspase 3
protease cleavage site. In other aspects of the embodiment, an
exogenous protease cleavage site located within the di-chain loop
region comprises, e.g., the consensus sequence
DX.sub.1X.sub.2D*X.sub.3 (SEQ ID NO: 515), where X.sub.1 is any
amino acid, with an acidic amino acid like D and E preferred,
X.sub.2 is any amino acid and X.sub.3 is amino acid, with a small
non-polar amino acid like A, C, G, S, and T preferred. In other
aspects of the embodiment, an exogenous protease cleavage site
located within the di-chain loop region comprises, e.g., SEQ ID NO:
516, SEQ ID NO: 517, SEQ ID NO: 518, SEQ ID NO: 519, SEQ ID NO:
520, or SEQ ID NO: 521.
[0347] A di-chain loop region can be modified so that a
naturally-occurring di-chain loop protease cleavage site is
replaced by an exogenous protease cleavage site. In this
modification, the naturally-occurring di-chain loop protease
cleavage site is made inoperable and thus cannot be cleaved by its
protease. Only the exogenous protease cleavage site can be cleaved
by its corresponding exogenous protease. In this type of
modification, the exogenous protease site is operably-linked to a
Clostridial toxin or Clostridial toxin chimeric as a fusion protein
and the site can be cleaved by its respective exogenous protease.
Replacement of an endogenous di-chain loop protease cleavage site
with an exogenous protease cleavage site can be a substitution of
the sites where the exogenous site is engineered at the position
approximating the cleavage site location of the endogenous site.
Replacement of an endogenous di-chain loop protease cleavage site
with an exogenous protease cleavage site can be an addition of an
exogenous site where the exogenous site is engineered at the
position different from the cleavage site location of the
endogenous site, the endogenous site being engineered to be
inoperable. The location and kind of protease cleavage site may be
critical because certain binding domains require a free
amino-terminal or carboxyl-terminal amino acid. For example, when a
peptide binding domain is placed between two other domains, e.g.,
see FIG. 4, a criterion for selection of a protease cleavage site
could be whether the protease that cleaves its site leaves a flush
cut, exposing the free amino-terminal or carboxyl-terminal of the
binding domain necessary for selective binding of the binding
domain to its receptor.
[0348] A naturally-occurring protease cleavage site can be made
inoperable by altering at least one of the two amino acids flanking
the peptide bond cleaved by the naturally-occurring di-chain loop
protease. More extensive alterations can be made, with the proviso
that the two cysteine residues of the di-chain loop region remain
intact and the region can still form the disulfide bridge.
Non-limiting examples of an amino acid alteration include deletion
of an amino acid or replacement of the original amino acid with a
different amino acid. Thus, in one embodiment, a
naturally-occurring protease cleavage site is made inoperable by
altering at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 amino acids
including at least one of the two amino acids flanking the peptide
bond cleaved by a naturally-occurring protease. In another
embodiment, a naturally-occurring protease cleavage site is made
inoperable by altering at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20 amino acids including at least one of the two amino acids
flanking the peptide bond cleaved by a naturally-occurring
protease.
[0349] It is understood that a modified Clostridial toxin disclosed
in the present specification can optionally further comprise a
flexible region comprising a flexible spacer. A flexible region
comprising flexible spacers can be used to adjust the length of a
polypeptide region in order to optimize a characteristic, attribute
or property of a polypeptide. As a non-limiting example, a
polypeptide region comprising one or more flexible spacers in
tandem can be used to better expose a protease cleavage site
thereby facilitating cleavage of that site by a protease. As
another non-limiting example, a polypeptide region comprising one
or more flexible spacers in tandem can be used to better present a
peptide binding domain, thereby facilitating the binding of that
binding domain to its receptor.
[0350] A flexible space comprising a peptide is at least one amino
acid in length and comprises non-charged amino acids with small
side-chain R groups, such as, e.g., small non-polar amino acids
like A, C, G, S, and T. Thus, in an embodiment a flexible spacer
can have a length of, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acids; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino
acids. In still another embodiment, a flexible spacer can be, e.g.,
between 1-3 amino acids, between 2-4 amino acids, between 3-5 amino
acids, between 4-6 amino acids, or between 5-7 amino acids.
Non-limiting examples of a flexible spacer include, e.g., a
G-spacers such as GGG, GGGG (SEQ ID NO: 522), and GGGGS (SEQ ID NO:
523) or an A-spacers such as AAA, AAAA (SEQ ID NO: 524) and AAAAT
(SEQ ID NO: 525). Such a flexible region is operably-linked
in-frame to the modified Clostridial toxin as a fusion protein.
[0351] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification can further
comprise a flexible region comprising a flexible spacer. In another
embodiment, a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification can further comprise
flexible region comprising a plurality of flexible spacers in
tandem. In aspects of this embodiment, a flexible region can
comprise in tandem, e.g., at least 1, 2, 3, 4, or 5 G-spacers; or
at most 1, 2, 3, 4, or 5 G-spacers. In still other aspects of this
embodiment, a flexible region can comprise in tandem, e.g., at
least 1, 2, 3, 4, or 5 A-spacers; or at most 1, 2, 3, 4, or 5
A-spacers. In another aspect of this embodiment, a Clostridial
toxin or Clostridial toxin chimeric can comprise a flexible region
comprising one or more copies of the same flexible spacers, one or
more copies of different flexible-spacer regions, or any
combination thereof.
[0352] It is envisioned that a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification can comprise
a flexible spacer in any and all locations with the proviso that
the Clostridial toxin or Clostridial toxin chimeric is capable of
performing the overall intoxication process. In aspects of this
embodiment, a flexible spacer is positioned between, e.g., an
enzymatic domain and a translocation domain, an enzymatic domain
and a binding domain, an enzymatic domain and an exogenous protease
cleavage site. In other aspects of this embodiment, a flexible
spacer is positioned between, e.g., a binding domain and a
translocation domain, a binding domain and an enzymatic domain, a
binding domain and an exogenous protease cleavage site. In yet
other aspects of this embodiment, a flexible spacer is positioned
between, e.g., a translocation domain and an enzymatic domain, a
translocation domain and a binding domain, a translocation domain
and an exogenous protease cleavage site.
[0353] As another non-limiting example of an optional component, a
Clostridial toxin or Clostridial toxin chimeric can further
comprise an epitope-binding region. An epitope-binding region can
be used in a wide variety of procedures involving, e.g., protein
purification and protein visualization. Such an epitope-binding
region is operably-linked in-frame to a modified Clostridial toxin
as a fusion protein. Non-limiting examples of an epitope-binding
region include, e.g., FLAG, Express.TM., human Influenza virus
hemagglutinin (HA), human p62.sup.c-Myc protein (c-MYC), Vesicular
Stomatitis Virus Glycoprotein (VSV-G), glycoprotein-D precursor of
Herpes simplex virus (HSV), V5, AU1, and AU5; affinity-binding,
such as. e.g., polyhistidine (HIS), streptavidin binding peptide
(strep), and biotin or a biotinylation sequence; peptide-binding
regions, such as. e.g., the glutathione binding domain of
glutathione-S-transferase, the calmodulin binding domain of the
calmodulin binding protein, and the maltose binding domain of the
maltose binding protein. Non-limiting examples of specific
protocols for selecting, making and using an appropriate binding
peptide are described in, e.g., Epitope Tagging, pp. 17.90-17.93
(Sambrook and Russell, eds., MOLECULAR CLONING A LABORATORY MANUAL,
Vol. 3, 3rd ed. 2001); ANTIBODIES: A LABORATORY MANUAL (Edward
Harlow & David Lane, eds., Cold Spring Harbor Laboratory Press,
2.sup.nd ed. 1998); and USING ANTIBODIES: A LABORATORY MANUAL:
PORTABLE PROTOCOL No. I (Edward Harlow & David Lane, Cold
Spring Harbor Laboratory Press, 1998). In addition, non-limiting
examples of binding peptides as well as well-characterized
reagents, conditions and protocols are readily available from
commercial vendors that include, without limitation, BD
Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen,
San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; QIAGEN, Inc.,
Valencia, Calif.; and Stratagene, La Jolla, Calif. These protocols
are routine procedures well within the scope of one skilled in the
art and from the teaching herein.
[0354] Thus, in an embodiment, a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification can further
comprise an epitope-binding region. In another embodiment, a
Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification can further comprises a plurality of
epitope-binding regions. In aspects of this embodiment, a
Clostridial toxin or Clostridial toxin chimeric can comprise, e.g.,
at least 1, 2, 3, 4, or 5 epitope-binding regions. In other aspects
of this embodiment, a Clostridial toxin or Clostridial toxin
chimeric can comprise, e.g., at most 1, 2, 3, 4, or 5
epitope-binding regions. In another aspect of this embodiment, a
modified Clostridial toxin can comprise one or more copies of the
same epitope-binding region, one or more copies of different
epitope-binding regions, or any combination thereof.
[0355] The location of an epitope-binding region can be in various
positions, including, without limitation, at the amino terminus,
within, or at the carboxyl terminus of a Clostridial toxin or
Clostridial toxin chimeric. Thus, in an embodiment, an
epitope-binding region is located at the amino-terminus of a
Clostridial toxin or Clostridial toxin chimeric. In another
embodiment, an epitope-binding region is located at the
carboxyl-terminus of a modified Clostridial toxin.
[0356] Aspects of the present specification provide, in part,
polynucleotide molecules. As used herein, the term "polynucleotide
molecule" is synonymous with "nucleic acid molecule" and refers to
a polymeric form of nucleotides, such as, e.g., ribonucleotides and
deoxyribonucleotides. It is envisioned that any and all
polynucleotide molecules that can encode a Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification
can be useful, including, without limitation naturally-occurring
and non-naturally-occurring DNA molecules and naturally-occurring
and non-naturally-occurring RNA molecules. Non-limiting examples of
naturally-occurring and non-naturally-occurring DNA molecules
include single-stranded DNA molecules, double-stranded DNA
molecules, genomic DNA molecules, cDNA molecules, vector
constructs, such as, e.g., plasmid constructs, phagemid constructs,
bacteriophage constructs, retroviral constructs and artificial
chromosome constructs. Non-limiting examples of naturally-occurring
and non-naturally-occurring RNA molecules include single-stranded
RNA, double stranded RNA and mRNA.
[0357] Well-established molecular biology techniques that may be
necessary to make a polynucleotide molecule encoding a Clostridial
toxin or Clostridial toxin chimeric disclosed in the present
specification including, but not limited to, procedures involving
polymerase chain reaction (PCR) amplification, restriction enzyme
reactions, agarose gel electrophoresis, nucleic acid ligation,
bacterial transformation, nucleic acid purification, nucleic acid
sequencing and recombination-based techniques are routine
procedures well within the scope of one skilled in the art and from
the teaching herein. Non-limiting examples of specific protocols
necessary to make a polynucleotide molecule encoding a modified
Clostridial toxin are described in e.g., MOLECULAR CLONING A
LABORATORY MANUAL, supra, (2001); and CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley
& Sons, 2004). Additionally, a variety of commercially
available products useful for making a polynucleotide molecule
encoding a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification are widely available. These
protocols are routine procedures well within the scope of one
skilled in the art and from the teaching herein.
[0358] Thus, in an embodiment, a polynucleotide molecule encodes a
Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification.
[0359] Another aspect of the present specification provides, in
part, a method of producing a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification, such method
comprising the step of expressing a polynucleotide molecule
encoding a Clostridial toxin or Clostridial toxin chimeric in a
cell. Another aspect of the present specification provides a method
of producing a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification, such method comprising the
steps of introducing an expression construct comprising a
polynucleotide molecule encoding a Clostridial toxin or Clostridial
toxin chimeric into a cell and expressing the expression construct
in the cell.
[0360] The methods disclosed in the present specification include,
in part, a Clostridial toxin or Clostridial toxin chimeric. It is
envisioned that any and all Clostridial toxins or Clostridial toxin
chimeras disclosed in the present specification can be produced
using the methods disclosed in the present specification. It is
also envisioned that any and all polynucleotide molecules encoding
a Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification can be useful in producing a Clostridial
toxin or Clostridial toxin chimeric disclosed in the present
specification using the methods disclosed in the present
specification.
[0361] The methods disclosed in the present specification include,
in part, an expression construct. An expression construct comprises
a polynucleotide molecule disclosed in the present specification
operably-linked to an expression vector useful for expressing the
polynucleotide molecule in a cell or cell-free extract. A wide
variety of expression vectors can be employed for expressing a
polynucleotide molecule encoding a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification, including,
without limitation, a viral expression vector; a prokaryotic
expression vector; eukaryotic expression vectors, such as, e.g., a
yeast expression vector, an insect expression vector and a
mammalian expression vector; and a cell-free extract expression
vector. It is further understood that expression vectors useful to
practice aspects of these methods may include those which express a
Clostridial toxin or Clostridial toxin chimeric under control of a
constitutive, tissue-specific, cell-specific or inducible promoter
element, enhancer element or both. Non-limiting examples of
expression vectors, along with well-established reagents and
conditions for making and using an expression construct from such
expression vectors are readily available from commercial vendors
that include, without limitation, BD Biosciences-Clontech, Palo
Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.;
Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences-Novagen,
Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La
Jolla, Calif. The selection, making and use of an appropriate
expression vector are routine procedures well within the scope of
one skilled in the art and from the teachings herein.
[0362] Thus, in aspects of this embodiment, a polynucleotide
molecule encoding a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification operably-linked to an
expression vector. In aspects of this embodiment, the expression
vector is, e.g., a viral expression vector, a prokaryotic
expression vector, a yeast expression vector, an insect expression
vector, or a mammalian expression vector. On other aspects of this
embodiment, a polynucleotide molecule encoding a Clostridial toxin
or Clostridial toxin chimeric disclosed in the present
specification operably-linked to a cell-free extract expression
vector.
[0363] The methods disclosed in the present specification include,
in part, a cell. It is envisioned that any and all cells can be
used. Thus, aspects of this embodiment include, without limitation,
prokaryotic cells including, without limitation, strains of
aerobic, microaerophilic, capnophilic, facultative, anaerobic,
gram-negative and gram-positive bacteria cells such as those
derived from, e.g., Escherichia coli, Bacillus subtilis, Bacillus
licheniformis, Bacteroides fragilis, Clostridia perfringens,
Clostridia difficile, Caulobacter crescentus, Lactococcus lactis,
Methylobacterium extorquens, Neisseria meningirulls, Neisseria
meningitidis, Pseudomonas fluorescens and Salmonella typhimurium;
and eukaryotic cells including, without limitation, yeast strains,
such as, e.g., those derived from Pichia pastoris, Pichia
methanolica, Pichia angusta, Schizosaccharomyces pombe,
Saccharomyces cerevisiae and Yarrowia lipolytica; insect cells and
cell lines derived from insects, such as, e.g., those derived from
Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and
Manduca sexta; and mammalian cells and cell lines derived from
mammalian cells, such as, e.g., those derived from mouse, rat,
hamster, porcine, bovine, equine, primate and human. Cell lines may
be obtained from the American Type Culture Collection, European
Collection of Cell Cultures and the German Collection of
Microorganisms and Cell Cultures. Non-limiting examples of specific
protocols for selecting, making and using an appropriate cell line
are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F.
A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES:
FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer
Academic Publishers, 1996); Maureen A. Harrison & Ian F. Rae,
GENERAL TECHNIQUES OF CELL CULTURE (Cambridge University Press,
1997); CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Alan Doyle
et al eds., John Wiley and Sons, 1998); R. Ian Freshney, CULTURE OF
ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (Wiley-Liss, 4th ed.
2000); ANIMAL CELL CULTURE: A PRACTICAL APPROACH (John R. W.
Masters ed., Oxford University Press, 3.sup.rd ed. 2000); MOLECULAR
CLONING A LABORATORY MANUAL, supra, (2001); BASIC CELL CULTURE: A
PRACTICAL APPROACH (John M. Davis, Oxford Press, 2nd ed. 2002); and
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). These
protocols are routine procedures within the scope of one skilled in
the art and from the teaching herein.
[0364] The methods disclosed in the present specification include,
in part, introducing into a cell a polynucleotide molecule. A
polynucleotide molecule introduced into a cell can be transiently
or stably maintained by that cell. Stably-maintained polynucleotide
molecules may be extra-chromosomal and replicate autonomously, or
they may be integrated into the chromosomal material of the cell
and replicate non-autonomously. It is envisioned that any and all
methods for introducing a polynucleotide molecule disclosed in the
present specification into a cell can be used. Methods useful for
introducing a polynucleotide molecule into a cell include, without
limitation, chemical-mediated transfection or transformation such
as, e.g., calcium chloride-mediated, calcium phosphate-mediated,
diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated,
polyethyleneimine (PEI)-mediated, polylysine-mediated and
polybrene-mediated; physical-mediated transfection or
transformation, such as, e.g., biolistic particle delivery,
microinjection, protoplast fusion and electroporation; and
viral-mediated transfection, such as, e.g., retroviral-mediated
transfection, see, e.g., Introducing Cloned Genes into Cultured
Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds.,
Molecular Cloning A Laboratory Manual, Vol. 3, 3.sup.rd ed. 2001).
One skilled in the art understands that selection of a specific
method to introduce an expression construct into a cell will
depend, in part, on whether the cell will transiently contain an
expression construct or whether the cell will stably contain an
expression construct. These protocols are routine procedures within
the scope of one skilled in the art and from the teaching
herein.
[0365] In an aspect of this embodiment, a chemical-mediated method,
termed transfection, is used to introduce into a cell a
polynucleotide molecule encoding a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification. In
chemical-mediated methods of transfection the chemical reagent
forms a complex with the nucleic acid that facilitates its uptake
into the cells. Such chemical reagents include, without limitation,
calcium phosphate-mediated, see, e.g., Martin Jordan & Florian
Worm, Transfection of adherent and suspended cells by calcium
phosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE)
dextran-mediated, lipid-mediated, cationic polymer-mediated like
polyethyleneimine (PEI)-mediated and polylysine-mediated and
polybrene-mediated, see, e.g., Chun Zhang et al., Polyethylenimine
strategies for plasmid delivery to brain-derived cells, 33(2)
Methods 144-150 (2004). Such chemical-mediated delivery systems can
be prepared by standard methods and are commercially available,
see, e.g., CellPhect Transfection Kit (Amersham Biosciences,
Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate
and DEAE Dextran, (Stratagene, Inc., La Jolla, Calif.);
LIPOFECTAMINE.TM. Transfection Reagent (Invitrogen, Inc., Carlsbad,
Calif.); ExGen 500 Transfection kit (Fermentas, Inc., Hanover,
Md.), and SuperFect and Effectene Transfection Kits (Qiagen, Inc.,
Valencia, Calif.).
[0366] In another aspect of this embodiment, a physical-mediated
method is used to introduce into a cell a polynucleotide molecule
encoding a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification. Physical techniques
include, without limitation, electroporation, biolistic and
microinjection. Biolistics and microinjection techniques perforate
the cell wall in order to introduce the nucleic acid molecule into
the cell, see, e.g., Jeike E. Biewenga et al., Plasmid-mediated
gene transfer in neurons using the biolistics technique, 71(1) J.
Neurosci. Methods 67-75 (1997); and John O'Brien & Sarah C. R.
Lummis, Biolistic and diolistic transfection: using the gene gun to
deliver DNA and lipophilic dyes into mammalian cells, 33(2) Methods
121-125 (2004). Electroporation, also termed
electropermeabilization, uses brief, high-voltage, electrical
pulses to create transient pores in the membrane through which the
nucleic acid molecules enter and can be used effectively for stable
and transient transfections of all cell types, see, e.g., M. Golzio
et al., In vitro and in vivo electric field-mediated
permeabilization, gene transfer, and expression, 33(2) Methods
126-135 (2004); and Oliver Gresch et al., New non-viral method for
gene transfer into primary cells, 33(2) Methods 151-163 (2004).
[0367] In another aspect of this embodiment, a viral-mediated
method, termed transduction, is used to introduce into a cell a
polynucleotide molecule encoding a Clostridial toxin or Clostridial
toxin chimeric disclosed in the present specification. In
viral-mediated methods of transient transduction, the process by
which viral particles infect and replicate in a host cell has been
manipulated in order to use this mechanism to introduce a
polynucleotide molecule into the cell. Viral-mediated methods have
been developed from a wide variety of viruses including, without
limitation, retroviruses, adenoviruses, adeno-associated viruses,
herpes simplex viruses, picornaviruses, alphaviruses and
baculoviruses, see, e.g., Armin Blesch, Lentiviral and MLV based
retroviral vectors for ex vivo and in vivo gene transfer, 33(2)
Methods 164-172 (2004); and Maurizio Federico, From lentiviruses to
lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M.
Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol.
Ther. 529-540 (2003); Karim Benihoud et al, Adenovirus vectors for
gene delivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H.
Bueler, Adeno-associated viral vectors for gene transfer and gene
therapy, 380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al.,
Adenovirus and adeno-associated virus vectors, 21(12) DNA Cell
Biol. 895-913 (2002); Edward A. Burton et al., Gene delivery using
herpes simplex virus vectors, 21(12) DNA Cell Biol. 915-936 (2002);
Paola Grandi et al., Targeting HSV amplicon vectors, 33(2) Methods
179-186 (2004); Ilya Frolov et al., Alphavirus-based expression
vectors: strategies and applications, 93(21) Proc. Natl. Acad. Sci.
U.S.A 11371-11377 (1996); Markus U. Ehrengruber, Alphaviral gene
transfer in neurobiology, 59(1) Brain Res. Bull. 13-22 (2002);
Thomas A. Kost & J. Patrick Condreay, Recombinant baculoviruses
as mammalian cell gene-delivery vectors, 20(4) Trends Biotechnol.
173-180 (2002); and A. Huser & C. Hofmann, Baculovirus vectors:
novel mammalian cell gene-delivery vehicles and their applications,
3(1) Am. J. Pharmacogenomics 53-63 (2003).
[0368] Adenoviruses, which are non-enveloped, double-stranded DNA
viruses, are often selected for mammalian cell transduction because
adenoviruses handle relatively large polynucleotide molecules of
about 36 kb, are produced at high titer, and can efficiently infect
a wide variety of both dividing and non-dividing cells, see, e.g.,
Wim T. J. M. C. Hermens et al., Transient gene transfer to neurons
and glia: analysis of adenoviral vector performance in the CNS and
PNS, 71(1) J. Neurosci. Methods 85-98 (1997); and Hiroyuki
Mizuguchi et al., Approaches for generating recombinant adenovirus
vectors, 52(3) Adv. Drug Deliv. Rev. 165-176 (2001). Transduction
using adenoviral-based system do not support prolonged protein
expression because the nucleic acid molecule is carried by an
episome in the cell nucleus, rather than being integrated into the
host cell chromosome. Adenoviral vector systems and specific
protocols for how to use such vectors are disclosed in, e.g.,
VIRAPOWER.TM. Adenoviral Expression System (Invitrogen, Inc.,
Carlsbad, Calif.) and VIRAPOWER.TM. Adenoviral Expression System
Instruction Manual 25-0543 version A, Invitrogen, Inc., (Jul. 15,
2002); and ADEASY.TM. Adenoviral Vector System (Stratagene, Inc.,
La Jolla, Calif.) and ADEASY.TM. Adenoviral Vector System
Instruction Manual 064004f, Stratagene, Inc.
[0369] Polynucleotide molecule delivery can also use
single-stranded RNA retroviruses, such as, e.g., oncoretroviruses
and lentiviruses. Retroviral-mediated transduction often produce
transduction efficiencies close to 100%, can easily control the
proviral copy number by varying the multiplicity of infection
(MOI), and can be used to either transiently or stably transduce
cells, see, e.g., Tiziana Tonini et al., Transient production of
retro viral- and lentiviral-based vectors for the transduction of
Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004); Armin
Blesch, Lentiviral and MLV based retroviral vectors for ex vivo and
in vivo gene transfer, 33(2) Methods 164-172 (2004); Felix
Recillas-Targa, Gene transfer and expression in mammalian cell
lines and transgenic animals, 267 Methods Mol. Biol. 417-433
(2004); and Roland Wolkowicz et al., Lentiviral vectors for the
delivery of DNA into mammalian cells, 246 Methods Mol. Biol.
391-411 (2004). Retroviral particles consist of an RNA genome
packaged in a protein capsid, surrounded by a lipid envelope. The
retrovirus infects a host cell by injecting its RNA into the
cytoplasm along with the reverse transcriptase enzyme. The RNA
template is then reverse transcribed into a linear, double stranded
cDNA that replicates itself by integrating into the host cell
genome. Viral particles are spread both vertically (from parent
cell to daughter cells via the provirus) as well as horizontally
(from cell to cell via virions). This replication strategy enables
long-term persistent expression since the nucleic acid molecules of
interest are stably integrated into a chromosome of the host cell,
thereby enabling long-term expression of the protein. For instance,
animal studies have shown that lentiviral vectors injected into a
variety of tissues produced sustained protein expression for more
than 1 year, see, e.g., Luigi Naldini et al., In vivo gene delivery
and stable transduction of non-dividing cells by a lentiviral
vector, 272(5259) Science 263-267 (1996). The
Oncoretroviruses-derived vector systems, such as, e.g., Moloney
murine leukemia virus (MoMLV), are widely used and infect many
different non-dividing cells. Lentiviruses can also infect many
different cell types, including dividing and non-dividing cells and
possess complex envelope proteins, which allows for highly specific
cellular targeting.
[0370] Retroviral vectors and specific protocols for how to use
such vectors are disclosed in, e.g., Manfred Gossen & Hermann
Bujard, Tight control of gene expression in eukaryotic cells by
tetracycline-responsive promoters, U.S. Pat. No. 5,464,758 (Nov. 7,
1995) and Hermann Bujard & Manfred Gossen, Methods for
regulating gene expression, U.S. Pat. No. 5,814,618 (Sep. 29, 1998)
David S. Hogness, Polynucleotides encoding insect steroid hormone
receptor polypeptides and cells transformed with same, U.S. Pat.
No. 5,514,578 (May 7, 1996) and David S. Hogness, Polynucleotide
encoding insect ecdysone receptor, U.S. Pat. No. 6,245,531 (Jun.
12, 2001); Elisabetta Vegeto et al., Progesterone receptor having
C. terminal hormone binding domain truncations, U.S. Pat. No.
5,364,791 (Nov. 15, 1994), Elisabetta Vegeto et al., Mutated
steroid hormone receptors, methods for their use and molecular
switch for gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23, 1999)
and Elisabetta Vegeto et al., Mutated steroid hormone receptors,
methods for their use and molecular switch for gene therapy, U.S.
Pat. No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral
delivery systems can be prepared by standard methods and are
commercially available, see, e.g., BD.TM. Tet-Off and Tet-On Gene
Expression Systems (BD Biosciences-Clontech, Palo Alto, Calif.) and
BD.TM. Tet-Off and Tet-On Gene Expression Systems User Manual,
PT3001-1, BD Biosciences Clontech, (Mar. 14, 2003), GENESWITCH.TM.
System (Invitrogen, Inc., Carlsbad, Calif.) and GENESWITCH.TM.
System A Mifepristone-Regulated Expression System for Mammalian
Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002);
VIRAPOWER.TM. Lentiviral Expression System (Invitrogen, Inc.,
Carlsbad, Calif.) and VIRAPOWER.TM. Lentiviral Expression System
Instruction Manual 25-0501 version E, Invitrogen, Inc., (Dec. 8,
2003); and COMPLETE CONTROL.RTM. Retroviral Inducible Mammalian
Expression System (Stratagene, La Jolla, Calif.) and COMPLETE
CONTROL.RTM. Retroviral Inducible Mammalian Expression System
Instruction Manual, 064005e.
[0371] The methods disclosed in the present specification include,
in part, expressing from a polynucleotide molecule a Clostridial
toxin or Clostridial toxin chimeric disclosed in the present
specification. It is envisioned that any of a variety of expression
systems may be useful for expressing from a polynucleotide molecule
a Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification, including, without limitation, cell-based
systems and cell-free expression systems. Cell-based systems
include, without limitation, viral expression systems, prokaryotic
expression systems, yeast expression systems, baculoviral
expression systems, insect expression systems, and mammalian
expression systems. Cell-free systems include, without limitation,
wheat germ extracts, rabbit reticulocyte extracts, and E. coli
extracts and generally are equivalent to the method disclosed
herein. Expression of a polynucleotide molecule using an expression
system can include any of a variety of characteristics including,
without limitation, inducible expression, non-inducible expression,
constitutive expression, viral-mediated expression,
stably-integrated expression, and transient expression. Expression
systems that include well-characterized vectors, reagents,
conditions and cells are well-established and are readily available
from commercial vendors that include, without limitation, Ambion,
Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.; BD
Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc,
Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied
Science, Indianapolis, Ind.; and Stratagene, La Jolla, Calif.
Non-limiting examples on the selection and use of appropriate
heterologous expression systems are described in e.g., PROTEIN
EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B. David Hames
eds., Oxford University Press, 1999); Joseph M. Fernandez &
James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE
ART OF EXPRESSION (Academic Press, 1999); and Meena Rai &
Harish Padh, Expression Systems for Production of Heterologous
Proteins, 80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols
are routine procedures well within the scope of one skilled in the
art and from the teaching herein.
[0372] A variety of cell-based expression procedures are useful for
expressing a polynucleotide molecule encoding a Clostridial toxin
or Clostridial toxin chimeric disclosed in the present
specification. Examples included, without limitation, viral
expression systems, prokaryotic expression systems, yeast
expression systems, baculoviral expression systems, insect
expression systems, and mammalian expression systems. Viral
expression systems include, without limitation, the VIRAPOWER.TM.
Lentiviral (Invitrogen, Inc., Carlsbad, Calif.), the Adenoviral
Expression Systems (Invitrogen, Inc., Carlsbad, Calif.), the
ADEASY.TM. XL Adenoviral Vector System (Stratagene, La Jolla,
Calif.) and the VIRAPORT.RTM. Retroviral Gene Expression System
(Stratagene, La Jolla, Calif.). Non-limiting examples of
prokaryotic expression systems include the CHAMPION.TM. pET
Expression System (EMD Biosciences-Novagen, Madison, Wis.), the
TRIEX.TM. Bacterial Expression System (EMD Biosciences-Novagen,
Madison, Wis.), the QIAEXPRESS.RTM. Expression System (QIAGEN,
Inc.), and the AFFINITY.RTM. Protein Expression and Purification
System (Stratagene, La Jolla, Calif.). Yeast expression systems
include, without limitation, the EASYSELECT.TM. Pichia Expression
Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-ECHO.TM.
Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and the
SPECTRA.TM. S. pombe Expression System (Invitrogen, Inc., Carlsbad,
Calif.). Non-limiting examples of baculoviral expression systems
include the BACULODIRECT.TM. (Invitrogen, Inc., Carlsbad, Calif.),
the BAC-TO-BAC.RTM. (Invitrogen, Inc., Carlsbad, Calif.), and the
BD BACULOGOLD.TM. (BD Biosciences-Pharmingen, San Diego, Calif.).
Insect expression systems include, without limitation, the
Drosophila Expression System (DES.RTM.) (Invitrogen, Inc.,
Carlsbad, Calif.), INSECTSELECT.TM. System (Invitrogen, Inc.,
Carlsbad, Calif.) and INSECTDIRECT.TM. System (EMD
Biosciences-Novagen, Madison, Wis.). Non-limiting examples of
mammalian expression systems include the T-REX.TM.
(Tetracycline-Regulated Expression) System (Invitrogen, Inc.,
Carlsbad, Calif.), the FLP-IN.TM. T-REX.TM. System (Invitrogen,
Inc., Carlsbad, Calif.), the pcDNA.TM. system (Invitrogen, Inc.,
Carlsbad, Calif.), the pSecTag2 system (Invitrogen, Inc., Carlsbad,
Calif.), the EXCHANGER.RTM. System, INTERPLAY.TM. Mammalian TAP
System (Stratagene, La Jolla, Calif.), COMPLETE CONTROL.RTM.
Inducible Mammalian Expression System (Stratagene, La Jolla,
Calif.) and LACSWITCH.RTM. II Inducible Mammalian Expression System
(Stratagene, La Jolla, Calif.).
[0373] Another procedure of expressing a polynucleotide molecule
encoding a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification employs a cell-free
expression system such as, without limitation, prokaryotic extracts
and eukaryotic extracts. Non-limiting examples of prokaryotic cell
extracts include the RTS 100 E. coli HY Kit (Roche Applied Science,
Indianapolis, Ind.), the ActivePro In Vitro Translation Kit
(Ambion, Inc., Austin, Tex.), the ECOPRO.TM. System (EMD
Biosciences-Novagen, Madison, Wis.) and the EXPRESSWAY.TM. Plus
Expression System (Invitrogen, Inc., Carlsbad, Calif.). Eukaryotic
cell extract include, without limitation, the RTS 100 Wheat Germ
CECF Kit (Roche Applied Science, Indianapolis, Ind.), the TNT.RTM.
Coupled Wheat Germ Extract Systems (Promega Corp., Madison, Wis.),
the Wheat Germ IVT.TM. Kit (Ambion, Inc., Austin, Tex.), the Retic
Lysate IVT.TM. Kit (Ambion, Inc., Austin, Tex.), the
PROTEINSCRIPT.RTM. II System (Ambion, Inc., Austin, Tex.) and the
TNT.RTM. Coupled Reticulocyte Lysate Systems (Promega Corp.,
Madison, Wis.).
[0374] The Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification disclosed in the present
specification are produced by the cell in a single-chain form. In
order to achieve full activity, this single-chain form has to be
converted into its di-chain form. As discussed above, this
conversion process is achieved by cleaving a protease cleavage site
located within the di-chain loop region of the Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification.
This conversion process can be performed using a standard in vitro
proteolytic cleavage assay or in a cell-based proteolytic cleavage
system as described in patent application Ghanshani, et al.,
Methods of Intracellular Conversion of Single-Chain Proteins into
their Di-chain Form, U.S. Pat. No. 8,546,108, which is hereby
incorporated by reference in its entirety.
[0375] Aspects of the present specification disclose, in part, a
composition comprising a Clostridial toxin or Clostridial toxin
chimeric disclosed in the present specification. In a further
aspect, the composition is a pharmaceutical acceptable composition.
As used herein, the term "pharmaceutically acceptable" refers to
any molecular entity or composition that does not produce an
adverse, allergic, or other untoward or unwanted reaction when
administered to an individual. As used herein, the term
"pharmaceutically acceptable composition" is synonymous with
"pharmaceutical composition" and refers to a therapeutically
effective concentration of an active ingredient, such as, e.g., any
of the Clostridial toxins or Clostridial toxin chimeras disclosed
in the present specification. A pharmaceutical composition
comprising a Clostridial toxin or Clostridial toxin chimeric is
useful for medical and veterinary applications. A pharmaceutical
composition may be administered to a patient alone, or in
combination with other supplementary active ingredients, agents,
drugs or hormones. The pharmaceutical compositions may be
manufactured using any of a variety of processes, including,
without limitation, conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
and lyophilizing. The pharmaceutical composition can take any of a
variety of forms including, without limitation, a sterile solution,
suspension, emulsion, lyophilizate, tablet, pill, pellet, capsule,
powder, syrup, elixir or any other dosage form suitable for
administration.
[0376] It is also envisioned that a pharmaceutical composition
comprising a Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification can optionally include a
pharmaceutically acceptable carriers that facilitate processing of
an active ingredient into pharmaceutically acceptable compositions.
As used herein, the term "pharmacologically acceptable carrier" is
synonymous with "pharmacological carrier" and refers to any carrier
that has substantially no long term or permanent detrimental effect
when administered and encompasses terms such as "pharmacologically
acceptable vehicle, stabilizer, diluent, additive, auxiliary, or
excipient." Such a carrier generally is mixed with an active
compound or is permitted to dilute or enclose the active compound
and can be a solid, semi-solid, or liquid agent. It is understood
that the active ingredients can be soluble or can be delivered as a
suspension in the desired carrier or diluent. Any of a variety of
pharmaceutically acceptable carriers can be used including, without
limitation, aqueous media such as, e.g., water, saline, glycine,
hyaluronic acid and the like; solid carriers such as, e.g.,
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like; solvents; dispersion media; coatings; antibacterial and
antifungal agents; isotonic and absorption delaying agents; or any
other inactive ingredient. Selection of a pharmacologically
acceptable carrier can depend on the mode of administration. Except
insofar as any pharmacologically acceptable carrier is incompatible
with the active ingredient, its use in pharmaceutically acceptable
compositions is contemplated. Non-limiting examples of specific
uses of such pharmaceutical carriers can be found in PHARMACEUTICAL
DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (Howard C. Ansel et al.,
eds., Lippincott Williams & Wilkins Publishers, 7th ed. 1999);
REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R. Gennaro
ed., Lippincott, Williams & Wilkins, 20th ed. 2000); GOODMAN
& GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Joel G.
Hardman et al., eds., McGraw-Hill Professional, 10th ed. 2001); and
HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhA
Publications, 4th edition 2003). These protocols are routine
procedures and any modifications are well within the scope of one
skilled in the art and from the teaching herein.
[0377] It is further envisioned that a pharmaceutical composition
disclosed in the present specification can optionally include,
without limitation, other pharmaceutically acceptable components
(or pharmaceutical components), including, without limitation,
buffers, preservatives, tonicity adjusters, salts, antioxidants,
osmolality adjusting agents, physiological substances,
pharmacological substances, bulking agents, emulsifying agents,
wetting agents, sweetening or flavoring agents, and the like.
Various buffers and refers to for adjusting pH can be used to
prepare a pharmaceutical composition disclosed in the present
specification, provided that the resulting preparation is
pharmaceutically acceptable. Such buffers include, without
limitation, acetate buffers, citrate buffers, phosphate buffers,
neutral buffered saline, phosphate buffered saline and borate
buffers. It is understood that acids or bases can be used to adjust
the pH of a composition as needed. Pharmaceutically acceptable
antioxidants include, without limitation, sodium metabisulfite,
sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and
butylated hydroxytoluene. Useful preservatives include, without
limitation, benzalkonium chloride, chlorobutanol, thimerosal,
phenylmercuric acetate, phenylmercuric nitrate, a stabilized oxy
chloro composition, such as, e.g., PURITE.RTM. and chelants, such
as, e.g., DTPA or DTPA-bisamide, calcium DTPA, and
CaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceutical
composition include, without limitation, salts such as, e.g.,
sodium chloride, potassium chloride, mannitol or glycerin and other
pharmaceutically acceptable tonicity adjustor. The pharmaceutical
composition may be provided as a salt and can be formed with many
different acids, including, but not limited to, hydrochloric,
sulfuric, acetic, lactic, tartaric, malic, and succinic. Salts tend
to be more soluble in aqueous or other protonic solvents than are
the corresponding free base forms. It is understood that these and
other substances known in the art of pharmacology can be included
in a pharmaceutical composition useful in the specification.
[0378] In an embodiment, a composition comprises a Clostridial
toxin or Clostridial toxin chimeric disclosed in the present
specification. In an aspect of this embodiment, the composition is
a pharmaceutical composition comprising a Clostridial toxin or
Clostridial toxin chimeric disclosed in the present specification.
In aspects of this embodiment, a pharmaceutical composition
comprising Clostridial toxin or Clostridial toxin chimeric
disclosed in the present specification further comprises a
pharmacological carrier, a pharmaceutical component, or both a
pharmacological carrier and a pharmaceutical component. In other
aspects of this embodiment, a pharmaceutical composition comprising
a Clostridial toxin or Clostridial toxin chimeric disclosed in the
present specification further comprises at least one
pharmacological carrier, at least one pharmaceutical component, or
at least one pharmacological carrier and at least one
pharmaceutical component.
[0379] Aspects of the present specification can also be described
as follows: [0380] 1. A Clostridial toxin comprising at least one
inactivation cleavage site located within an inactivation cleavage
site region, wherein the inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain, wherein the at least one inactivation cleavage site
comprises a dual Thrombin-Thrombin site, a Factor Xa site, a dual
Factor Xa-Thrombin site, and/or a MMP-9 site. [0381] 2. A
Clostridial toxin comprising a Clostridial toxin enzymatic domain,
a Clostridial toxin translocation domain, a Clostridial toxin
binding domain, a di-chain loop region, an exogenous protease
cleavage site, and at least two inactivation cleavage sites located
within an inactivation cleavage site region; wherein the exogenous
protease cleavage site located within the di-chain loop region.
[0382] 3. A Clostridial toxin of aspect 2, wherein the inactivation
cleavage sites comprise a dual Thrombin-Thrombin site and/or a dual
Factor Xa-Thrombin site. [0383] 4. A Clostridial toxin chimeric
comprising a Clostridial toxin enzymatic domain, a Clostridial
toxin translocation domain, a non-Clostridial toxin binding domain,
and an inactivation cleavage located within an inactivation
cleavage site region, wherein the inactivation cleavage site region
is located in the translocation domain and/or the H.sub.CN binding
subdomain. [0384] 5. The Clostridial toxin and/or Clostridial toxin
chimeric of aspects 1-4, wherein the inactivation cleavage site
region comprises amino acids 462-496 of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 4 and/or SEQ ID NO: 5; amino acids 458-492 of SEQ ID NO:
3; amino acids 464-487 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, and/or SEQ ID NO: 10; amino acids 463-496 of SEQ ID
NO: 11 and/or SEQ ID NO: 12; amino acids 458-491 of SEQ ID NO: 13
and/or SEQ ID NO: 14; amino acids 434-467 of SEQ ID NO: 15, SEQ ID
NO: 16, and/or SEQ ID NO: 17; amino acids 453-486 of SEQ ID NO: 18,
SEQ ID NO: 19, and/or SEQ ID NO: 20; amino acids 458-491 of SEQ ID
NO: 21; amino acids 443-476 of SEQ ID NO: 23; and/or amino acids
434-467 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0385] 6. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects 1-4,
wherein the inactivation cleavage site region comprises amino acids
618-634 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID
NO: 5; amino acids 614-630 of SEQ ID NO: 3; amino acids 605-621 of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ
ID NO: 10; amino acids 613-629 of SEQ ID NO: 11 and/or SEQ ID NO:
12; amino acids 609-625 of SEQ ID NO: 13 and/or SEQ ID NO: 14;
amino acids 587-603 of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID
NO: 17; amino acids 604-620 of SEQ ID NO: 18; amino acids 605-621
of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids 610-626 of SEQ
ID NO: 21; amino acids 596-612 of SEQ ID NO: 23; and/or amino acids
587-603 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0386] 7. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects 1-4,
wherein the inactivation cleavage site region comprises amino acids
638-651 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID
NO: 5; amino acids 634-647 of SEQ ID NO: 3; amino acids 625-638 of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ
ID NO: 10; amino acids 633-646 of SEQ ID NO: 11 and/or SEQ ID NO:
12; amino acids 629-642 of SEQ ID NO: 13 and/or SEQ ID NO: 14;
amino acids 607-620 of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID
NO: 17; amino acids 624-637 of SEQ ID NO: 18; amino acids 625-638
of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids 630-643 of SEQ
ID NO: 21; amino acids 616-629 of SEQ ID NO: 23; and/or amino acids
607-620 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0387] 8. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects 1-4,
wherein the inactivation cleavage site region comprises amino acids
665-687 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID
NO: 5; amino acids 661-683 of SEQ ID NO: 3; amino acids 652-674 of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ
ID NO: 10; amino acids 660-682 of SEQ ID NO: 11 and/or SEQ ID NO:
12; amino acids 656-678 of SEQ ID NO: 13 and/or SEQ ID NO: 14;
amino acids 634-659 of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID
NO: 17; amino acids 651-676 of SEQ ID NO: 18; amino acids 652-677
of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids 657-679 of SEQ
ID NO: 21; amino acids 643-668 of SEQ ID NO: 23; and/or amino acids
634-659 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0388] 9. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects 1-4,
wherein the inactivation cleavage site region comprises amino acids
752-765 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID
NO: 5; amino acids 748-761 of SEQ ID NO: 3; amino acids 739-752 of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ
ID NO: 10; amino acids 747-760 of SEQ ID NO: 11 and/or SEQ ID NO:
12; amino acids 743-756 of SEQ ID NO: 13 and/or SEQ ID NO: 14;
amino acids 724-739 of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID
NO: 17; amino acids 741-756 of SEQ ID NO: 18; amino acids 742-757
of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids 744-757 of SEQ
ID NO: 21; amino acids 733-748 of SEQ ID NO: 23; and/or amino acids
724-739 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0389] 10. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects 1-4,
wherein the inactivation cleavage site region comprises amino acids
826-835 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID
NO: 5; amino acids 824-831 of SEQ ID NO: 3; amino acids 813-824 of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ
ID NO: 10; amino acids 821-830 of SEQ ID NO: 11 and/or SEQ ID NO:
12; amino acids 817-826 of SEQ ID NO: 13 and/or SEQ ID NO: 14;
amino acids 800-809 of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID
NO: 17; amino acids 817-826 of SEQ ID NO: 18; amino acids 818-827
of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids 818-827 of SEQ
ID NO: 21; amino acids 809-819 of SEQ ID NO: 23; and/or amino acids
800-809 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0390] 11. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects 1-4,
wherein the inactivation cleavage site region comprises amino acids
844-863 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID
NO: 5; amino acids 840-859 of SEQ ID NO: 3; amino acids 831-850 of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ
ID NO: 10; amino acids 839-858 of SEQ ID NO: 11 and/or SEQ ID NO:
12; amino acids 835-854 of SEQ ID NO: 13 and/or SEQ ID NO: 14;
amino acids 818-837 of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID
NO: 17; amino acids 835-854 of SEQ ID NO: 18; amino acids 836-855
of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids 836-855 of SEQ
ID NO: 21; amino acids 828-847 of SEQ ID NO: 23; and/or amino acids
818-837 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0391] 12. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects 1-4,
wherein the inactivation cleavage site region comprises amino acids
871-895 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID
NO: 5; amino acids 867-891 of SEQ ID NO: 3; amino acids 858-882 of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ
ID NO: 10; amino acids 866-890 of SEQ ID NO: 11 and/or SEQ ID NO:
12; amino acids 862-886 of SEQ ID NO: 13 and/or SEQ ID NO: 14;
amino acids 845-869 of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID
NO: 17; amino acids 862-886 of SEQ ID NO: 18; amino acids 863-887
of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids 863-887 of SEQ
ID NO: 21; amino acids 855-879 of SEQ ID NO: 23; and/or amino acids
845-869 of SEQ ID NO: 24 and/or SEQ ID NO: 25. [0392] 13. The
Clostridial toxin and/or Clostridial toxin chimeric of aspects
1-12, wherein the Clostridial toxin enzymatic domain comprises a
BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1
enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic
domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a
BaNT enzymatic domain, and/or a BuNT enzymatic domain. [0393] 14.
The Clostridial toxin and/or Clostridial toxin chimeric of aspects
1-13, wherein the inactivation cleavage site comprises Thrombin
cleavage sites, Plasmin cleavage sites, Coagulation Factor VIIa
cleavage sites, Coagulation Factor IXa cleavage sites, Coagulation
Factor Xa cleavage sites, Coagulation Factor XIa cleavage sites,
Coagulation Factor XIIa cleavage sites, plasma kallikrein cleavage
sites, protease-activated G protein-coupled receptor-1 (PAR1)
cleavage sites, PAR2 cleavage sites, PAR3 cleavage sites, PAR4
cleavage sites, Matrix Metalloproteinase-2 (MMP-2) cleavage sites,
Matrix Metalloproteinase-9 (MMP-9) cleavage sites, Furin cleavage
sites, urokinase-type Plasminogen activator (uPA) cleavage sites,
tissue-type Plasminogen activator (tPA) cleavage sites,
Tryptase-.epsilon. cleavage sites, Mouse mast cell protease-7
(mMCP-7) cleavage sites, endothelin-converting enzyme-1 (ECE-1)
cleavage sites, Kell blood group cleavage sites, DPPIV cleavage
sites, ADAM metallopeptidase with thrombospondin type 1 motif-13
(ADAMTS13) cleavage sites, and/or Cathepsin L cleavage sites.
[0394] 15. The Clostridial toxin and/or Clostridial toxin chimeric
of aspects 1-14, wherein the Clostridial toxin translocation domain
comprises a BoNT/A translocation domain, a BoNT/B translocation
domain, a BoNT/C1 translocation domain, a BoNT/D translocation
domain, a BoNT/E translocation domain, a BoNT/F translocation
domain, a BoNT/G translocation domain, a TeNT translocation domain,
a BaNT translocation domain, and/or a BuNT translocation domain.
[0395] 16. The Clostridial toxin and/or Clostridial toxin chimeric
of aspects 1-15, wherein the inactivation cleavage site comprises a
dual Thrombin-Thrombin site, a Factor Xa site, a dual Factor
Xa-Thrombin site, and/or a MMP-9 site. [0396] 17. The Clostridial
toxin and/or Clostridial toxin chimeric of aspects 1-16, wherein
the, a non-Clostridial toxin binding domain, comprises a opioid
binding domain, a tachykinin binding domain, a melanocortin binding
domain, a galanin binding domain, a granin binding domain, a
Neuropeptide Y related peptide binding domain, a neurohormone
binding domain, a neuroregulatory cytokine binding domain, a kinin
peptide binding domain, a growth factor binding domain, and/or a
glucagon like hormone binding domain. [0397] 18. A BoNT/A
comprising an inactivation cleavage site located within an
inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0398] 19. A Clostridial toxin
comprising a BoNT/A enzymatic domain, a BoNT/A translocation
domain, a BoNT/A binding domain, and an inactivation cleavage site
located within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0399] 20. A
Clostridial toxin comprising a BoNT/A enzymatic domain, a BoNT/A
translocation domain, a BoNT/A binding domain, an exogenous
protease cleavage site, a di-chain loop region, and an inactivation
cleavage site located within an inactivation cleavage site region,
wherein the exogenous protease cleavage site is located within the
di-chain loop region; the wherein inactivation cleavage site region
is located in the translocation domain and/or the H.sub.CN binding
subdomain. [0400] 21. A Clostridial toxin comprising a BoNT/A
enzymatic domain, a BoNT/A translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0401] 22. A
Clostridial toxin comprising a BoNT/A enzymatic domain, a BoNT/A
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0402] 23. The toxin and/or
chimeric of aspects 18-22, wherein the inactivation cleavage site
region comprises amino acids 462-496, 618-634, 638-651, 665-687,
752-765, 826-835, 844-863, and/or 871-895 of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 4 and/or SEQ ID NO: 5, and/or amino acids
458-492, 614-630, 634-647, 665-687, 748-761, 822-831, 840-859,
and/or 867-891 of SEQ ID NO: 3. [0403] 24. A BoNT/B comprising an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain. [0404] 25. A Clostridial toxin comprising a BoNT/B
enzymatic domain, a BoNT/B translocation domain, a BoNT/B binding
domain, and an inactivation cleavage site located within an
inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0405] 26. A Clostridial toxin
comprising a BoNT/B enzymatic domain, a BoNT/B translocation
domain, a BoNT/B binding domain, an exogenous protease cleavage
site, a di-chain loop region, and an inactivation cleavage site
located within an inactivation cleavage site region, wherein the
exogenous protease cleavage site is located within the di-chain
loop region; the wherein inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain. [0406] 27. A Clostridial toxin comprising a BoNT/B
enzymatic domain, a BoNT/B translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0407] 28. A
Clostridial toxin comprising a BoNT/B enzymatic domain, a BoNT/B
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0408] 29. The toxin and/or
chimeric of aspects 24-28, wherein the inactivation cleavage site
region comprises amino acids 464-487, 605-621, 625-638, 652-674,
739-752, 813-824, 831-850, and/or 858-882 of SEQ ID NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ ID NO: 10. [0409] 30.
A BoNT/C1 comprising an inactivation cleavage site located within
an inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H
.sub.CN binding subdomain. [0410] 31. A Clostridial toxin
comprising a BoNT/C1 enzymatic domain, a BoNT/C1 translocation
domain, a BoNT/C1 binding domain, and an inactivation cleavage site
located within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0411] 32. A
Clostridial toxin comprising a BoNT/C1 enzymatic domain, a BoNT/C1
translocation domain, a BoNT/C1 binding domain, an exogenous
protease cleavage site, a di-chain loop region, and an inactivation
cleavage site located within an inactivation cleavage site region,
wherein the exogenous protease cleavage site is located within the
di-chain loop region; the wherein inactivation cleavage site region
is located in the translocation domain and/or the H.sub.CN binding
subdomain. [0412] 33. A Clostridial toxin comprising a BoNT/C1
enzymatic domain, a BoNT/C1 translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0413] 34. A
Clostridial toxin comprising a BoNT/C1 enzymatic domain, a BoNT/C1
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0414] 35. The toxin and/or
chimeric of aspects 30-34, wherein the inactivation cleavage site
region comprises amino acids 463-496, 613-629, 633-646, 660-682,
747-760, 821-830, 839-858, and/or 866-890 of SEQ ID NO: 11 and/or
SEQ ID NO: 12. [0415] 36. A BoNT/D comprising an inactivation
cleavage site located within an inactivation cleavage site region,
wherein the inactivation cleavage site region is located in the
translocation domain and/or the H.sub.CN binding subdomain. [0416]
37. A Clostridial toxin comprising a BoNT/D enzymatic domain, a
BoNT/D translocation domain, a BoNT/D binding domain, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain. [0417] 38. A Clostridial toxin comprising a BoNT/D
enzymatic domain, a BoNT/D translocation domain, a BoNT/D binding
domain, an exogenous protease cleavage site, a di-chain loop
region, and an inactivation cleavage site located within an
inactivation cleavage site region, wherein the exogenous protease
cleavage site is located within the di-chain loop region; the
wherein inactivation cleavage site region is located in the
translocation domain and/or the H.sub.CN binding subdomain. [0418]
39. A Clostridial toxin comprising a BoNT/D enzymatic domain, a
BoNT/D translocation domain, a non-Clostridial toxin binding
domain, and an inactivation cleavage site located within an
inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0419] 40. A Clostridial toxin
comprising a BoNT/D enzymatic domain, a BoNT/D translocation
domain, a non-Clostridial toxin binding domain, an exogenous
protease cleavage site, a di-chain loop region, and an inactivation
cleavage site located within an inactivation cleavage site region,
wherein the exogenous protease cleavage site is located within the
di-chain loop region; the wherein inactivation cleavage site region
is located in the translocation domain and/or the H.sub.CN binding
subdomain. [0420] 41. The toxin and/or chimeric of aspects 36-40,
wherein the inactivation cleavage site region comprises amino acids
458-491, 609-625, 629-642, 656-678, 743-756, 817-826, 835-854,
and/or 862-886 of SEQ ID NO: 13 and/or SEQ ID NO: 14. [0421] 42. A
BoNT/E comprising an inactivation cleavage site located within an
inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0422] 43. A Clostridial toxin
comprising a BoNT/E enzymatic domain, a BoNT/E translocation
domain, a BoNT/E binding domain, and an inactivation cleavage site
located within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0423] 44. A
Clostridial toxin comprising a BoNT/E enzymatic domain, a BoNT/E
translocation domain, a BoNT/E binding domain, an exogenous
protease cleavage site, a di-chain loop region, and an inactivation
cleavage site located within an inactivation cleavage site region,
wherein the exogenous protease cleavage site is located within the
di-chain loop region; the wherein inactivation cleavage site region
is located in the translocation domain and/or the H.sub.CN binding
subdomain. [0424] 45. A Clostridial toxin comprising a BoNT/E
enzymatic domain, a BoNT/E translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0425] 46. A
Clostridial toxin comprising a BoNT/E enzymatic domain, a BoNT/E
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0426] 47. The toxin and/or
chimeric of aspects 42-46, wherein the inactivation cleavage site
region comprises amino acids 434-467, 587-603, 607-620, 634-659,
724-739, 800-809, 818-837, and/or 845-869 of SEQ ID NO: 15, SEQ ID
NO: 16, and/or SEQ ID NO: 17. [0427] 48. A BoNT/F comprising an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain. [0428] 49. A Clostridial toxin comprising a BoNT/F
enzymatic domain, a BoNT/F translocation domain, a BoNT/F binding
domain, and an inactivation cleavage site located within an
inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0429] 50. A Clostridial toxin
comprising a BoNT/F enzymatic domain, a BoNT/F translocation
domain, a BoNT/F binding domain, an exogenous protease cleavage
site, a di-chain loop region, and an inactivation cleavage site
located within an inactivation cleavage site region, wherein the
exogenous protease cleavage site is located within the di-chain
loop region; the wherein inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain. [0430] 51. A Clostridial toxin comprising a BoNT/F
enzymatic domain, a BoNT/F translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0431] 52. A
Clostridial toxin comprising a BoNT/F enzymatic domain, a BoNT/F
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0432] 53. The toxin and/or
chimeric of aspects 48-52, wherein the inactivation cleavage site
region comprises amino acids 453-486, 604-620, 624-637, 651-676,
741-756, 817-826, 835-854, and/or 862-886 of SEQ ID NO: 18; and/or
amino acids 453-486, 605-621, 625-638, 652-677, 742-757, 818-827,
836-855, and/or 863-887 of SEQ ID NO: 19 and/or SEQ ID NO: 20.
[0433] 54. A BoNT/G comprising an inactivation cleavage site
located within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0434] 55. A
Clostridial toxin comprising a BoNT/G enzymatic domain, a BoNT/G
translocation domain, a BoNT/G binding domain, and an inactivation
cleavage site located within an inactivation cleavage site region,
wherein the inactivation cleavage site region is located in the
translocation domain and/or the H.sub.CN binding subdomain. [0435]
56. A Clostridial toxin comprising a BoNT/G enzymatic domain, a
BoNT/G translocation domain, a BoNT/G binding domain, an exogenous
protease cleavage site, a di-chain loop region, and an inactivation
cleavage site located within an inactivation cleavage site region,
wherein the exogenous protease cleavage site is located within the
di-chain loop region; the wherein inactivation cleavage site region
is located in the translocation domain and/or the H.sub.CN binding
subdomain. [0436] 57. A Clostridial toxin comprising a BoNT/G
enzymatic domain, a BoNT/G translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0437] 58. A
Clostridial toxin comprising a BoNT/G enzymatic domain, a BoNT/G
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0438] 59. The toxin and/or
chimeric of aspects 54-58, wherein the inactivation cleavage site
region comprises amino acids 458-491, 610-626, 630-643, 657-679,
744-757, 818-827, 836-855, and/or 863-887 of SEQ ID NO: 21. [0439]
60. A BaNT comprising an inactivation cleavage site located within
an inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0440] 61. A Clostridial toxin
comprising a BaNT enzymatic domain, a BaNT translocation domain, a
BaNT binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0441] 62. A
Clostridial toxin comprising a BaNT enzymatic domain, a BaNT
translocation domain, a BaNT binding domain, an exogenous protease
cleavage site, a di-chain loop region, and an inactivation cleavage
site located within an inactivation cleavage site region, wherein
the exogenous protease cleavage site is located within the di-chain
loop region; the wherein inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain. [0442] 63. A Clostridial toxin comprising a BaNT
enzymatic domain, a BaNT translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0443] 64. A
Clostridial toxin comprising a BaNT enzymatic domain, a BaNT
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0444] 65. The toxin and/or
chimeric of aspects 60-64, wherein the inactivation cleavage site
region comprises amino acids 443-476, 596-612, 616-629, 643-668,
733-748, 809-819, 828-847, and/or 855-879 of SEQ ID NO: 23. [0445]
66. A BuNT comprising an inactivation cleavage site located within
an inactivation cleavage site region, wherein the inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0446] 67. A Clostridial toxin
comprising a BuNT enzymatic domain, a BuNT translocation domain, a
BuNT binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0447] 68. A
Clostridial toxin comprising a BuNT enzymatic domain, a BuNT
translocation domain, a BuNT binding domain, an exogenous protease
cleavage site, a di-chain loop region, and an inactivation cleavage
site located within an inactivation cleavage site region, wherein
the exogenous protease cleavage site is located within the di-chain
loop region; the wherein inactivation cleavage site region is
located in the translocation domain and/or the H.sub.CN binding
subdomain. [0448] 69. A Clostridial toxin comprising a BuNT
enzymatic domain, a BuNT translocation domain, a non-Clostridial
toxin binding domain, and an inactivation cleavage site located
within an inactivation cleavage site region, wherein the
inactivation cleavage site region is located in the translocation
domain and/or the H.sub.CN binding subdomain. [0449] 70. A
Clostridial toxin comprising a BuNT enzymatic domain, a BuNT
translocation domain, a non-Clostridial toxin binding domain, an
exogenous protease cleavage site, a di-chain loop region, and an
inactivation cleavage site located within an inactivation cleavage
site region, wherein the exogenous protease cleavage site is
located within the di-chain loop region; the wherein inactivation
cleavage site region is located in the translocation domain and/or
the H.sub.CN binding subdomain. [0450] 71. The toxin and/or
chimeric of aspects 66-70, wherein the inactivation cleavage site
region comprises amino acids 434-467, 587-603, 607-620, 634-659,
724-739, 800-809, 818-837, and/or 845-869 of SEQ ID NO: 24 and/or
SEQ ID NO: 25. [0451] 72. The toxin and/or chimeric of aspects
1-71, wherein the Clostridial toxin and/or Clostridial toxin
chimeric comprising an inactivation cleavage site has a safety
margin that is greater relative to the same and/or similar to the
Clostridial toxin and/or Clostridial toxin chimeric, but without
the inactivation cleavage site.
[0452] 73. The toxin and/or chimeric of aspect 72, wherein the
Clostridial toxin and/or Clostridial toxin chimeric comprising an
inactivation cleavage site has a safety margin that is greater than
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 100%, 110%, at least 120%, at least 130%, at least 140%, at
least 150%, at least 160%, at least 170%, at least 180%, at least
190%, at least 200%, 210%, at least 220%, at least 230%, at least
240%, at least 250%, at least 260%, at least 270%, at least 280%,
at least 290%, and/or at least 300%, relative to the same and/or
similar Clostridial toxin and/or Clostridial toxin chimeric, but
without the inactivation cleavage site, and/or [0453] wherein
Clostridial toxin and/or Clostridial toxin chimeric comprising an
inactivation cleavage site has a safety margin that is greater than
at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at
most 60%, at most 70%, at most 80%, at most 90%, at most 100%,
110%, at most 120%, at most 130%, at most 140%, at most 150%, at
most 160%, at most 170%, at most 180%, at most 190%, at most 200%,
210%, at most 220%, at most 230%, at most 240%, at most 250%, at
most 260%, at most 270%, at most 280%, at most 290%, and/or at most
300%, relative to the same and/or similar Clostridial toxin and/or
Clostridial toxin chimeric, but without the inactivation cleavage
site, and/or [0454] wherein the Clostridial toxin and/or
Clostridial toxin chimeric comprising an inactivation cleavage site
has a safety margin that is greater by about 10% to about 300%,
about 20% to about 300%, about 30% to about 300%, about 40% to
about 300%, about 50% to about 300%, about 60% to about 300%, about
70% to about 300%, about 80% to about 300%, about 90% to about
300%, and/or about 100% to about 300%, relative to the same and/or
similar Clostridial toxin and/or Clostridial toxin chimeric, but
without the inactivation cleavage site, or [0455] wherein the
Clostridial toxin and/or Clostridial toxin chimeric comprising an
inactivation cleavage site has a safety margin that is greater than
at least 1-fold, at least 1-fold, at least 3-fold, at least 4-fold,
at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold,
at least 9-fold, and/or at least 10-fold, relative to the same
and/or similar Clostridial toxin and/or Clostridial toxin chimeric,
but without the inactivation cleavage site, and/or [0456] wherein
the Clostridial toxin and/or Clostridial toxin chimeric comprising
an inactivation cleavage site has a safety margin that is greater
than at least 1-fold, at most 1-fold, at most 3-fold, at most
4-fold, at most 5-fold, at most 6-fold, at most 7-fold, at most
8-fold, at most 9-fold, and/or at most 10-fold, relative to the
same and/or similar Clostridial toxin and/or Clostridial toxin
chimeric, but without the inactivation cleavage site, and/or [0457]
wherein the Clostridial toxin and/or Clostridial toxin chimeric
comprising an inactivation cleavage site has a safety margin that
is greater by about 1-fold to about 10-fold, about 1-fold to about
9-fold, about 1-fold to about 8-fold, about 1-fold to about 7-fold,
about 1-fold to about 6-fold, about 1-fold to about 5-fold, about
2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold
to about 8-fold, about 2-fold to about 7-fold, about 2-fold to
about 6-fold, and/or about 2-fold to about 5-fold. [0458] 74. The
toxin and/or chimeric of aspects 1-73, wherein the addition of the
inactivation cleavage site increases the safety margin of the
Clostridial toxin and/or Clostridial toxin chimeric relative to the
same and/or similar Clostridial toxin and/or Clostridial toxin
chimeric, but without the additional inactivation cleavage site.
[0459] 75. The toxin and/or chimeric of aspect 74, wherein the
Clostridial toxin and/or Clostridial toxin chimeric comprises the
addition of an inactivation cleavage site that increases the safety
margin of the Clostridial toxin and/or Clostridial toxin chimeric
relative to the same and/or similar Clostridial toxin and/or
Clostridial toxin chimeric, but without the additional inactivation
cleavage site by at least 10%, at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, 110%, at least 120%, at least 130%, at
least 140%, at least 150%, at least 160%, at least 170%, at least
180%, at least 190%, at least 200%, 210%, at least 220%, at least
230%, at least 240%, at least 250%, at least 260%, at least 270%,
at least 280%, at least 290%, and/or at least 300%, or [0460]
wherein the Clostridial toxin and/or Clostridial toxin chimeric
comprises the addition of an inactivation cleavage site that
increases the safety margin of the Clostridial toxin and/or
Clostridial toxin chimeric relative to the same and/or similar
Clostridial toxin and/or Clostridial toxin chimeric, but without
the additional inactivation cleavage site by at most 10%, at most
20%, at most 30%, at most 40%, at most 50%, at most 60%, at most
70%, at most 80%, at most 90%, at most 100%, 110%, at most 120%, at
most 130%, at most 140%, at most 150%, at most 160%, at most 170%,
at most 180%, at most 190%, at most 200%, 210%, at most 220%, at
most 230%, at most 240%, at most 250%, at most 260%, at most 270%,
at most 280%, at most 290%, and/or at most 300%, or [0461] wherein
the Clostridial toxin and/or Clostridial toxin chimeric comprises
the addition of an inactivation cleavage site that increases the
safety margin of the Clostridial toxin and/or Clostridial toxin
chimeric relative to the same and/or similar Clostridial toxin
and/or Clostridial toxin chimeric, but without the additional
inactivation cleavage site by about 10% to about 300%, about 20% to
about 300%, about 30% to about 300%, about 40% to about 300%, about
50% to about 300%, about 60% to about 300%, about 70% to about
300%, about 80% to about 300%, about 90% to about 300%, and/or
about 100% to about 300%, or [0462] wherein the Clostridial toxin
and/or Clostridial toxin chimeric comprises the addition of an
inactivation cleavage site that increases the safety margin of the
Clostridial toxin and/or Clostridial toxin chimeric relative to the
same and/or similar Clostridial toxin and/or Clostridial toxin
chimeric, but without the additional inactivation cleavage site by
at least 1-fold, at least 1-fold, at least 3-fold, at least 4-fold,
at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold,
at least 9-fold, and/or at least 10-fold, and/or [0463] wherein the
Clostridial toxin and/or Clostridial toxin chimeric comprises the
addition of an inactivation cleavage site that increases the safety
margin of the Clostridial toxin and/or Clostridial toxin chimeric
relative to the same and/or similar Clostridial toxin and/or
Clostridial toxin chimeric, but without the additional inactivation
cleavage site by, e.g., at most 1-fold, at most 3-fold, at most
4-fold, at most 5-fold, at most 6-fold, at most 7-fold, at most
8-fold, at most 9-fold, and/or at most 10-fold, and/or wherein the
Clostridial toxin and/or Clostridial toxin chimeric comprises the
addition of an inactivation cleavage site that increases the safety
margin of the Clostridial toxin and/or Clostridial toxin chimeric
relative to the same and/or similar Clostridial toxin and/or
Clostridial toxin chimeric, but without the additional inactivation
cleavage site by about 1-fold to about 10-fold, about 1-fold to
about 9-fold, about 1-fold to about 8-fold, about 1-fold to about
7-fold, about 1-fold to about 6-fold, about 1-fold to about 5-fold,
about 2-fold to about 10-fold, about 2-fold to about 9-fold, about
2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold
to about 6-fold, and/or about 2-fold to about 5-fold. [0464] 76. A
polynucleotide molecule encoding a toxin and/or chimeric according
to any one of aspects 1-75. [0465] 77, The polynucleotide molecule,
wherein the molecule comprises SEQ ID NO: 530, SEQ ID NO: 532, SEQ
ID NO: 534, and/or SEQ ID NO: 536. [0466] 78. A method of producing
a Clostridial toxin and/or Clostridial toxin chimeric comprising
the step of expressing in a cell a polynucleotide molecule
according to aspect 76 and/or 77, wherein expression from the
polynucleotide molecule produces the encoded Clostridial toxin
and/or Clostridial toxin chimeric. [0467] 79. A method of producing
a modified Clostridial toxin comprising the steps of: [0468] a.
introducing into a cell a polynucleotide molecule according to
aspect 76 and/or 77; and [0469] b. expressing the polynucleotide
molecule, wherein expression from the polynucleotide molecule
produces the encoded Clostridial toxin and/or Clostridial toxin
chimeric. [0470] 80. A Clostridial toxin comprising SEQ ID NO: 531,
SEQ ID NO: 533, SEQ ID NO: 535, and/or SEQ ID NO: 537. [0471] 81. A
Clostridial toxin comprising SEQ ID NO: 531. [0472] 82. A
Clostridial toxin comprising SEQ ID NO: 533. [0473] 83. A
Clostridial toxin comprising SEQ ID NO: 535. [0474] 84. A
Clostridial toxin comprising SEQ ID NO: 537.
EXAMPLES
[0475] The following non-limiting examples are provided for
illustrative purposes only in order to facilitate a more complete
understanding of disclosed embodiments and are in no way intended
to limit any of the embodiments disclosed in the present
specification.
Example 1
Identification of Inactivation Cleavage Site Regions
[0476] This example illustrates how to identify regions within a
Clostridial toxin or Clostridial toxin chimeric suitable for
modifying the toxin to comprise an inactivation cleavage site and
how to make a Clostridial toxin or Clostridial toxin chimeric
comprising an inactivation cleavage site.
[0477] To identify a location or locations in the protein structure
suitable as a potential inactivation cleavage site region, the
three-dimensional structure of a BoNT/A was initially analyzed by
computer software to identify surface exposed loops or extended
regions that would be more accessible to a protease. Of the regions
predicted to be accessible, eight were selected for further
analysis: amino acids 462-496 of SEQ ID NO: 1, amino acids 618-634
of SEQ ID NO: 1, amino acids 638-651 of SEQ ID NO: 1, amino acids
665-687 of SEQ ID NO: 1, amino acids 752-765 of SEQ ID NO: 1, and
amino acids 826-835 of SEQ ID NO: 1, amino acids 844-863 of SEQ ID
NO: 1, and amino acids 871-895 of SEQ ID NO: 1.
[0478] To determine whether a region identified by computer
analysis could function as an inactivation cleavage site region,
thrombin cleavage sites were genetically engineered into these
regions using multi-primer mutagenesis and assayed for its ability
to be cleaved by thrombin. A 50 .mu.L reaction was assembled
comprising a primer pool of unidirectional oligonucleotide primers
each containing the desired modification (125 ng of each primer)
mixed in different ratios with a DNA template comprising an
expression construct encoding a BoNT/A, such as, e.g., an
expression construct comprising SEQ ID NO: 526 encoding SEQ ID NO:
527, or an expression construct comprising SEQ ID NO: 528 encoding
SEQ ID NO: 529, that was hypermethylated with dam methylase. To
this mixture was added 5 .mu.L of 10.times.PCR Buffer, 1 .mu.L of
deoxyribonucleotides (dNTPs), 1 .mu.L of 2.5 units/.mu.L
PFUULTRA.TM. High Fidelity DNA polymerase (Stratagene, La Jolla,
Calif.), Pfu DNA ligase, ATP, and nuclease-free water to a final
volume of 50 .mu.L. The thermocycler conditions were: 30 cycles of
96.degree. C. for 1 minute, 60.degree. C. for 30 seconds, and
68.degree. C. for 20 minutes. Following thermocycling, 1 .mu.L of
DpnI restriction enzyme (Stratagene, La Jolla, Calif.) was added to
the reaction and incubated for 1 hour at 37.degree. C. to digest
the template DNA and reduce the recovery of wild-type clones. The
digested reaction mixture was transformed into electro-competent E.
coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by
electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0)
containing 50 .mu.g/mL of kanamycin, and placed in a 37.degree. C.
incubator for overnight growth. Bacteria containing expression
constructs were identified as kanamycin resistant colonies.
Candidate constructs were isolated using an alkaline lysis plasmid
mini-preparation procedure and analyzed by sequencing to determine
the frequency and identity of the mutations incorporated. Table 7
lists each BoNT/A comprising a Thrombin cleavage site (BoNT/A-TCS)
made and tested in this thrombin scanning analysis.
TABLE-US-00007 TABLE 7 Thrombin Scanning Analysis Thrombin BoNT/A
Region Modification Expression Sensitivity Potency 462-496
T482insLVPRGS + ND ND 462-496 A489insLVPRGS ++ ++ ND 618-634
E620insLVPRGS + ND ND 638-651 M646insLVPRGS -/+ ND ND 665-687
I673insLVPRGS + ND ND 752-765 E758insLVPRGS - ND ND 826-835
delR827GT-insLVPRGS -/+ ND ND 844-863 T844insLVPRGS +++ + ND
844-863 D848insLVPRGS +++ + ND 844-863 Q852insLVPRGS -/+ ND ND
844-863 L862insLVPRGS +++ ++ ND 871-895 E868insLVPRGS ND 871-895
delE868YIKNI-insLVPRGS ND 871-895 K871insLVPRGS +++ +++ ND 871-895
I873insLVPRGS +++ ++++ ND 871-895 delN872IINTS-insLVPRGS ND 871-895
T876insLVPRGS ND 871-895 L879insVPRGS ND 871-895
delL879NLRYE-insLVPRGS ND 871-895 N880insLVPRGS +++ ++++ 4.05
871-895 L881insVPRGS ND 871-895 delL881RYESN-insLVPRGS ND 871-895
Y883insLVPRGS ND 871-895 E884insLVPRGS +++ +++ >50 871-895
S885insLVPRGS ND 871-895 delH887LIDLS-insLVPRGS ND 871-895
L888insVPRGS ND 871-895 D890insLVPRGS ++ ++++ 3.15 871-895
L891insVPRG ND 871-895 delS892RYA-insVPRG ND 467-496 T482insLVPRGS
+ ND ND A489insLVPRGS 618-634 E620insLVPRGS + ND ND 665-687
I673insLVPRGS 638-651 M646insLVPRGS + ND ND 665-687 I673insLVPRGS
825-832 delR827GT-insLVPRGS + +++ ND 871-895 K871insLVPRGS 844-863
T844insLVPRGS -/+ ND ND Q852insLVPRGS 844-863 Q852insLVPRGS - ND ND
L862insLVPRGS 825-832 delR827GT-insLVPRGS + ND ND 871-895
K871insLVPRGS 825-832 delR827GT-insLVPRGS - ND ND 871-895
K880insLVPRGS 871-895 E868insLVPRGS 1.32 I873insLVPRGS 871-895
delE868YIKNI-insLVPRGS 0.86 delL881RYESN-insLVPRGS 871-895
I873insLVPRGS 1.32 E884insLVPRGS 871-895 L881insVPRGS +++ ++++ 4.20
L891insVPRG Control Backbone +++ - Yes Protease sensitivity: +,
less than 25% of toxin proteolyzed within about 1 to about 4 hours;
++, from 25% to 50% of toxin proteolyzed within about 1 to about 4
hours; +++, from 51% to 75% of toxin proteolyzed within about 1 to
about 4 hours; ++++, more than 75% of toxin proteolyzed within
about 1 to about 4 hours. BoNT/A potency is calculated by dividing
the EC.sub.50 value of the toxin into the EC.sub.50 value of the
backbone control. ND is not determined.
[0479] To determine the expression level of soluble protein for
each BoNT/A-TCS, an expression construct comprising each BoNT/A-TCS
was expressed, purified by immobilized metal affinity
chromatography and analyzed by SDS-PAGE analysis. First, using a
96-well plate, 100 .mu.L of PA-0.5G media containing 50 .mu.g/mL
Kanamycin was inoculated with a single colony of BL21(DE3) cells
harboring the appropriate expression construct and grown at
37.degree. C. with shaking overnight. A 5 .mu.L aliquot from this
starter culture was used to inoculate 1 mL of ZYP-5052 containing
50 .mu.g/mL kanamycin and grown at 37.degree. C. with shaking for
3.5 hours and then 22.degree. C. for 16 hours. A 110 .mu.L aliquot
of Protein Extraction Reagent comprising 10.times. FASTBREAK.TM.
Cell Lysis Reagent (Promega Corp., Madison, Wis.), 250 U/mL
Benzonase nuclease (EMD Biosciences-Novagen, Madison, Wis.), and
10.times. Protease Inhibitor Cocktail III (EMD
Biosciences-Calbiochem, Gibbstown, N.J.) was added to each 1 mL
expression culture in a 96-well plate. 75 .mu.L of HISLINK.TM.
resin (Promega Corp., Madison, Wis.) was next transferred to each
well and the mixture was alternately mixed by pipetting and by
shaking at 900 rpm for 30 minutes. The lysates were transferred to
a filter plate with a 25 .mu.m pore size (Promega Corp., Madison,
Wis.), with membranes pre-wetted with water, and the liquid was
removed by vacuum filtration. The resin was washed three times with
200 .mu.L Wash Buffer comprising 100 mM HEPES (pH 7.5), 10 mM
imidazole. The protein was eluted by adding 200 .mu.L Elution
Buffer comprising 100 mM HEPES (pH 7.5), 500 mM imidazole,
incubating for 5 minutes and the elute collected by vacuum
filtration into a 96-well plate.
[0480] To perform SDS-PAGE, an equal volume of 2.times. Laemmli
Sample Buffer was added to the IMAC purified BoNT/A comprising a
thrombin cleavage site, and the mixture incubated at 95.degree. C.
for 5 minutes. A 15 .mu.L aliquot was loaded and separated by MOPS
polyacrylamide gel electrophoresis using NUPAGE.RTM. Novex 4-12%
Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad,
Calif.) under denaturing, reducing conditions. The gel was washed
and fixed in 10% methanol and 7% acetic acid for 30 minutes. The
wash solution was removed and the gel incubated in SYPRO Ruby
protein gel stain solution for 3 hours to overnight at room
temperature. The stained gel was destained in 10% methanol and 7%
acetic acid for 30 minutes. The destained gel was visualized with a
Fluro-S-Max digital imager (Bio-Rad).
[0481] The results of the expression analysis are given in Table 7.
In general, toxins harboring an inserted thrombin cleavage site in
the inactivation regions comprising amino acids 462-496 of SEQ ID
NO: 1, amino acids 844-863, or amino acids 871-895 of SEQ ID NO: 1
were expressed well. For example, toxins comprising A489insLVPRGS
was expressed at about 50% that of a wild-type control construct
and toxins comprising D848insLVPRGS or N880insLVPRGS were expressed
at, or near, control levels (Table 7). These results reveal that
inactivation cleavage site regions located within the translocation
domain and/or the H.sub.CN binding subdomain tolerated the
modification of regions to include a protease cleavage site.
[0482] To further explore the extent to which the inactivation
cleavage site regions identified could tolerate modifications that
introduce a protease cleavage site, toxins were modified to include
thrombin cleavage sites throughout the region. For example, toxins
comprising T884insLVPRGS or L862ins LVPRGS were made to examine the
inactivation cleavage site region comprising 844-863 of SEQ ID NO:
1. Similarly, toxins comprising E868insLVPRGS,
delE868YIKNI-insLVPRGS, delN8721INTS-insLVPRGS, T876insLVPRGS,
L879insVPRGS, delL879NLRYE-insLVPRGS, L881 insVPRGS, delL881
RYESN-insLVPRGS, Y883insLVPRGS, E884insLVPRGS, S885insLVPRGS,
delH887LIDLS-insLVPRGS, L888insLVPRGS, L891insVPRG, and
delS892RYA-insVPRG were made to examine the inactivation cleavage
site region comprising 871-895 of SEQ ID NO: 1. Both insertion and
substitution modifications were made to examine whether the type of
modification had any affect. In general, all toxins harboring an
inserted thrombin cleavage site in these inactivation regions were
expressed at, or near, the levels of a wild-type control construct.
These results reveal that inactivation cleavage site regions within
the translocation domain and/or the H.sub.CN binding subdomain can
tolerate modifications placed anywhere within an inactivation site
region.
[0483] Lastly, the ability of an inactivation site region to
tolerate the presence of two or more protease cleavage sites was
examined (Table 7). These results indicate that inactivation
cleavage site regions within the translocation domain and/or the
H.sub.CN binding subdomain can tolerate modifications placing two
or more protease cleavage sites within an inactivation site
region.
[0484] To determine whether a BoNT/A comprising a thrombin cleavage
site could be cleaved by thrombin, an in vitro thrombin cleavage
assay was performed. 5 .mu.g of each purified BoNT/A-TCS was
incubated with 1 U of Thrombin (Novagen) at 23.degree. C. for 1
hour, 3 hours, and 18.5 hours. A zero-enzyme control was also set
up in parallel for each BoNT/A-TCS. Samples were taken at each time
point and quenched with SDS-Loading Buffer including DTT and
analyzed by SDS-PAGE as described above.
[0485] The results of the expression analysis are given in Table 7.
In general, modification of an inactivation cleavage site region
comprising amino acids 467-496, 844-863, or 871-895 of SEQ ID NO: 1
to include a protease cleavage site resulted in a toxin that was
susceptible to proteolytic cleavage by the appropriate
protease.
[0486] To determine whether a BoNT/A comprising a thrombin cleavage
site maintained its potency, a BoNT/A activity assay was performed
using a cell-based activity assay. To conduct a cell-based activity
assay, about 1.2.times.10.sup.6 Neuro-2a or SiMa cells were plated
into the wells of 24-well tissue culture plates containing 1 mL of
serum-free medium containing Minimum Essential Medium, 2 mM
GLUTAMAX.TM. I with Earle's salts, 1.times.B27 supplement,
1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM
HEPES and 25 .mu.g/mL GT1b. The cells were incubated in a
37.degree. C. incubator under 5% carbon dioxide until the cells
differentiated, as assessed by standard and routine morphological
criteria, such as growth arrest and neurite extension
(approximately 3 days). The media was aspirated from each well and
replaced with either 1) fresh media containing no toxin (untreated
cell line) or 2) fresh media containing 1 nM of a BoNT/A complex
(treated cell line). After an overnight incubation, the cells were
washed by aspirating the media and rinsing each well with 200 .mu.L
of 1.times.PBS. To harvest the cells, the 1.times.PBS was
aspirated, the cells were lysed by adding 50 .mu.l of 2.times.SDS
Loading Buffer, the lysate was transferred to a clean test tube and
the sample was heated to 95.degree. C. for 5 minutes.
[0487] To detect for the presence of cleaved SNAP-25 products, an
aliquot from each harvested sample was analyzed by Western blot. In
this analysis, a 12 .mu.l aliquot of the harvested sample was
separated by MOPS polyacrylamide gel electrophoresis using
NUPAGE.RTM. Novex 12% Bis-Tris precast polyacrylamide gels
(Invitrogen Inc., Carlsbad, Calif.) under denaturing, reducing
conditions. Separated peptides were transferred from the gel onto
polyvinylidene fluoride (PVDF) membranes (Invitrogen Inc.,
Carlsbad, Calif.) by Western blotting using a TRANS-BLOT.RTM. SD
semi-dry electrophoretic transfer cell apparatus (Bio-Rad
Laboratories, Hercules, Calif.). PVDF membranes were blocked by
incubating at room temperature for 2 hours in a solution containing
Tris-Buffered Saline (TBS) (25 mM
2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid
(Tris-HCl)(pH 7.4), 137 mM sodium chloride, 2.7 mM potassium
chloride), 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaurate), 2% Bovine Serum Albumin (BSA), 5% nonfat dry milk.
Blocked membranes were incubated at 4.degree. C. for overnight in
TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaurate), 2% BSA, and 5% nonfat dry milk containing either 1) a
1:5,000 dilution of an .alpha.-SNAP-25 mouse monoclonal antibody as
the primary antibody (SMI-81; Sternberger Monoclonals Inc.,
Lutherville, Md.); or 2) a 1:5,000 dilution of S9684
.alpha.-SNAP-25 rabbit polyclonal antiserum as the primary antibody
(Sigma, St. Louis, Mo.). Both .alpha.-SNAP-25 mouse monoclonal and
rabbit polyclonal antibodies can detect both the uncleaved SNAP-25
substrate and the SNAP-25 cleavage product, allowing for the
assessment of overall SNAP-25 expression in each cell line and the
percent of SNAP-25 cleaved after BoNT/A treatment as a parameter to
assess the amount of BoNT/A uptake. Primary antibody probed blots
were washed three times for 15 minutes each time in TBS,
TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaurate). Washed
membranes were incubated at room temperature for 2 hours in TBS,
0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaurate), 2%
BSA, and 5% nonfat dry milk containing either 1) a 1:10,000
dilution of goat polyclonal anti-mouse immunoglobulin G, heavy and
light chains (IgG, H+L) antibody conjugated to horseradish
peroxidase (Zymed, South San Francisco, Calif.) as a secondary
antibody; or 2) a 1:10,000 dilution of goat polyclonal anti-rabbit
immunoglobulin G, heavy and light chains (IgG, H+L) antibody
conjugated to horseradish peroxidase (Zymed, South San Francisco,
Calif.) as a secondary antibody. Secondary antibody-probed blots
were washed three times for 15 minutes each time in TBS, 0.1%
TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaurate). Signal
detection of the labeled SNAP-25 products were visualized using the
ECL Plus.TM. Western Blot Detection System (GE Healthcare, Amersham
Biosciences, Piscataway, N.J.) and the membrane was imaged and the
percent of cleaved quantified with a Typhoon 9410 Variable Mode
Imager and Imager Analysis software (GE Healthcare, Amersham
Biosciences, Piscataway, N.J.). The choice of pixel size (100 to
200 pixels) and PMT voltage settings (350 to 600, normally 400)
depended on the individual blot.
[0488] The results of the expression analysis are given in Table 7.
In general, modification of an inactivation cleavage site region
comprising amino acids 467-496, 844-863, or 871-895 of SEQ ID NO: 1
to include a protease cleavage site resulted in a potent toxin that
was able to execute the overall intoxication process.
[0489] Taken together, these results indicate that although eight
different inactivation cleavage regions were identified, not all
were able to support the insertion of a functional thrombin
cleavage site. In general, modification of the inactivation
cleavage site regions comprising amino acids 467-496, 844-863 and
871-895 of SEQ ID NO: 1 to include a protease cleavage site
resulted in a stably produced toxin that was able to execute the
overall intoxication process and was sensitive to proteolytic
cleavage by the appropriate protease.
[0490] Because the three-dimensional structure of all Clostridial
toxins are similar, the corresponding locations in BoNT/B, BoNT/C1,
BoNT/D, BoNT/E, BoNT/F, BoNT/G, TeNT, BaNT, and BuNT are also
suitable as inactivation cleavage site regions. Table 5 lists these
regions.
Example 2
Protease Cleavage Site Analysis
[0491] This example illustrates how to make a Clostridial toxin or
Clostridial toxin chimeric comprising an inactivation cleavage
site.
[0492] To explore whether protease cleavage sites other than
thrombin could be useful as an inactivation site, toxins comprising
many different protease cleavage sites were examined.
[0493] To make a Clostridial toxin or Clostridial toxin chimeric
comprising an inactivation cleavage site, protease cleavage sites
were genetically engineered into inactivation cleavage site regions
using multi-primer mutagenesis as described in Example 1. Table 8
lists the expression constructs modified to contain a protease
cleavage site.
[0494] To determine whether a BoNT/A comprising a protease cleavage
site could be cleaved by its cognate protease, in vitro protease
cleavage assays was performed essentially as described above, but
using the appropriate protease instead of thrombin. Samples were
taken at each time point and quenched with SDS-Loading Buffer
including DTT, and analyzed by SDS-PAGE as described in Example
1.
[0495] The results of the expression analysis are given in Table 7.
In general, modification of an inactivation cleavage site region
comprising amino acids 467-496, 844-863, or 871-895 of SEQ ID NO: 1
to include a protease cleavage site resulted in a toxin that was
susceptible to proteolytic cleavage by the appropriate
protease.
TABLE-US-00008 TABLE 8 Protease Cleavage Site Analysis Protease
Protease BoNT/A Cleavage Site Region Modification Sensitivity
Potency Factor Xa 535 E535insG + 2.70 Factor Xa 844-863 L863insIEGR
+ >50 Factor Xa 871-895 K871insIEGR ++ 6.15 Factor Xa 871-895
I873insEGR + 3.97 Factor Xa 871-895 L881insIEG ND ND Factor Xa
871-895 E884insIEGR + 2.95 Factor Xa 871-895 L891insIEGR ++ ND
Factor Xa 1272 E1272insG + ND Factor Xa x 2 535 E535insG + ND 1272
E1272insG Factor Xa x 2 871-895 K871insIEGR ++ 4.35 L891insIEGR
Factor Xa x 2 871-895 I873insEGR + 7.63 L891insIEGR Factor Xa x 2
871-895 L881insIEG ++ >50 L891insIEGR Factor Xa 871-895
I873insEGR ND ND tPA delS885NHLIDL-insPQRGRSA Factor Xa 871-895
I873insEGR + 3.29 Thrombin E884insLVPRG ++++ MMP-2 871-895
S885insGPLGMLSQ + 6.55 MMP-2 871-895 delK871NIINTSI-insGPLGMLSQ ++
5.27 MMP-2 871-895 delS885NHLIDLS-insGPLGMLSQ ++ 4.76 MMP-9 871-895
K871insGPLGLWAQ ND ND MMP-9 871-895 delK871NIINTSI-insGPLGLWAQ +
3.36 MMP-9 871-895 I873insGPLGLWAQ 22.8 MMP-9 871-895
delI874NTSILNL-insGPLGLWAQ 37.7 MMP-9 871-895
delL881RYESNHL-insGPLGLWAQ ND ND MMP-9 871-895 E884insGPLGLWAQ ND
ND MMP-9 871-895 delS885NHLIDLS-insGPLGLWAQ + 4.38 MMP-9 871-895
S885insGPLGLWAQ 3.38 MMP-9 871-895 L891insGPLGLWAQ 20.61 MMP-9
871-895 delK871NIINTSI-insGPLGLWAQ ND ND Thrombin E884insLVPRG
MMP-9 871-895 delK871NIINTSI-insGPLGLWAQ 19.62 Factor Xa
E884insIEGR u-PA 871-895 delN872IINTSI-insPGSGKSA + ND u-PA 871-895
S885insPGSGKSA ++ 3.00 u-PA 871-895 delN886HLIDL-insPGSGKSA ++ 4.90
t-PA 871-895 delN872IINTSI-insPQRGRSA ++ 3.65 t-PA 871-895
S885insPQRGRSA +++ 3.30 t-PA 871-895 delS885NHLIDL-insPQRGRSA ++
4.80 Thrombin 871-895 I873LVPRGS ND ND tPA delS885NHLIDL-insPQRGRSA
Furin 871-895 I870insRKKR +++ 6.70 Furin 871-895 delK871NII-insRKKR
+ 3.50 Furin 871-895 L881insRKK + 7.20 Furin 871-895
delY883ES-insKKR + 12.1 Furin 871-895 S892RKK + 15.2 Furin x 2
871-895 delK871NII-insRKKR + 12.6 delY883ES-insKKR Furin x 2
871-895 delK871NII-insRKKR ++ 6.00 S892RKK Furin x 3 871-895
delK871NII-insRKKR ND ND delY883ES-insKKR S892RKK Kell 871-895
L891insAAF + 10.8 Kell 871-895 delI889DL-insAAF + 4.80 Tryptase
.epsilon. 871-895 K871insIVGGE + 9.45 Tryptase .epsilon. 871-895
K871insRIVGGE + 6.48 Tryptase .epsilon. 871-895
delN886HLIDL-insRIVGGE 5.50 Tryptase .epsilon. 871-895
delN886HLIDL-insKIVGGE ND ND mMMCP-7 871-895 K871insSLSSRQSP 3.90
mMMCP-7 871-895 delN886HLIDLS-insLSSRQSP 4.80 ECE-1 871-895
I870insRPPGFSAF + 5.70 ECE-1 871-895 K871insAFA + 3.85 ECE-1
871-895 K871insDIIWVNTPEHVVPYGLGS + >50 ECE-1 871-895
K871insRPKPQQFFGLM ND ND ECE-1 871-895 delYES885NHLIDLS- + 9.20
insPKPQQFFGLM ECE-1 871-895 E884insKAFA + 2.95 ECE-1 871-895
delS885NHLIDLS-insRPPGFSAF + 3.70 Cathespin L 871-895
I870insRGFFYTPK ++++ 10.3 Cathespin L 871-895 K871insLR ++++ 2.25
Cathespin L 871-895 K871insFR ++++ 3.05 Cathespin L 871-895
K871insLR 12.6 Thrombin L891insLVPRGS PolyArg 844-863 R861insRR ND
ND PolyArg 871-895 R882insRRR Yes PolyArg 871-895 S885insRRR 2.22
PolyArg 871-895 S892insRRR 3.02 PolyArg x 2 844-863 R861insRR ND ND
871-895 K871insRKR PolyArg x 2 844-863 R861insRR ND ND 871-895
I873insRRRR PolyArg x 2 844-863 R861insRR ND ND 871-895 R882insRRR
PolyArg x 2 871-895 K871insRKR 1.92 S885insRRR PolyArg x 2 871-895
R882insRRR ND ND S892insRRR Protease sensitivity: +, less than 25%
of toxin proteolyzed within about 1 to about 4 hours; ++, from 25%
to 50% of toxin proteolyzed within about 1 to about 4 hours; +++,
from 51% to 75% of toxin proteolyzed within about 1 to about 4
hours; ++++, more than 75% of toxin proteolyzed within about 1 to
about 4 hours. BoNT/A potency is calculated by dividing the
EC.sub.50 value of the toxin into the EC.sub.50 value of the
backbone control. ND is not determined.
[0496] To determine whether a BoNT/A comprising a protease cleavage
site maintained its potency, the cell-based activity assay
described above was performed (Table 8). In general, toxins
comprising a protease cleavage site that exhibited an EC.sub.50 of
about 20 or less were deemed to retain enough potency to warranted
evaluation using an animal-based assay.
Example 3
In Vivo Analysis
[0497] This example illustrates how to evaluate a Clostridial toxin
or Clostridial toxin chimeric comprising an inactivation cleavage
site using an animal-based assay analysis.
[0498] Although the cell-based activity assay is a good assessment
of whether a Clostridial toxin or Clostridial toxin chimeric
comprising an inactivation cleavage site can be cleaved by its
cognate protease, certain candidates were selected for evaluation
in an animal-based assay.
[0499] To test the activity of a Clostridial toxin or Clostridial
toxin chimeric comprising an inactivation cleavage site using an
animal-based assay, an in vivo Digit Abduction Score (DAS) assay
was initially performed. CD-1 Fe mice were weighed and placed into
subsets of 10 animals for each discrete DAS assay. Mice were
included into a particular subset based on the following criteria:
1) good health; 2) robust baseline DAS response of 0; 3) inclusion
in a median weight range of X.+-.2 g established for the selected
subset and 4) weight greater than 17.0 g.
[0500] Each mouse was injected using a 30-gauge needle in the
gastrocnemius muscle of the right hind limb with either 1) 5 .mu.L
of 10.0 nM BoNT/A comprising an inactivation cleavage site
(single-dose DAS study); or 2) 5 .mu.L of one of seven different
doses of BoNT/A comprising an inactivation cleavage site (0.01 nM,
0.04 nM, 0.12 nM, 0.37 nM, 1.11 nM, 3.33 nM and 10.0 nM;
Full-Dosing DAS study). As a control, the gastrocnemius muscle of
the left hind limb was injected with 5 .mu.L of a solution not
containing any toxin. Mice were observed for the DAS response
consecutively for the first 4 days. The DAS was read by lifting
each mouse by the tail and precisely observing the injected hind
limbs. The abduction or no abduction of the hind digits reveals the
effect of paralysis due to the test toxin injected in the muscle.
The digit abduction of the injected hind limb was compared with
that of the non-injected hind limb and scored accordingly. DAS data
was analyzed by calculating the ED.sub.50 dose based on peak mean
DAS score and AUC (area under the curve) in terms of u/Kg and/or
ng/Kg. This was accomplished as follows: 1) the mean peak DAS score
for each dose was calculated in each study; 2) any dose that
elicited more than five deaths in any study was eliminated from
consideration; 3) the highest dose used in a given individual study
was the lowest dose which elicited an average peak of 4.0; 4) the
lowest dose used in a given individual study was the highest dose
which elicited an average peak of 0; 5) curves were constructed for
each individual study of average peak DAS vs. log (dose); 6) an AUC
value was calculated for each group of 10 mice of the multiple
groups in some studies; 7) curves were constructed for each
individual study of average AUC vs. log (dose); 8) an x, y
replicate response curve was constructed for each set of multiple
identical studies; for each test toxin; 9) dose-response data were
analyzed by non-linear regression (non-weighted) using a
three-parameter logistic equation (Sigma Plot v 8.0; SPSS Science,
Chicago, Ill.) using the following equation:
y=a/(1+(x/x0).sup.b)
where y is the response, a is the asymptotic y.sub.max, b is the
slope, x is the dose, and 0 is the ED.sub.50 dose, For peak
ED.sub.50 determinations, Y.sub.max was set to 4 (maximum DAS
reading on scale). Mean (peak and/or AUC) ED.sub.50 values were
computed for each eight-dose study performed.
[0501] The results indicate that (Table 9). In general, toxins
comprising an inactivation cleavage site that exhibited a relative
potency of about 10 or above were deemed to retain enough potency
to warranted evaluation of its safety margin.
[0502] To determine the safety margin of a Clostridial toxin or
Clostridial toxin chimeric comprising an inactivation cleavage
site, a mouse lethality assay was performed.
[0503] To calculate the safety margin of a Clostridial toxin or
Clostridial toxin chimeric comprising an inactivation cleavage
site, the LD.sub.50 value obtained from the mouse lethality assay
was divided by the EC.sub.50 value obtained from a full-dosing DAS
study. A toxin comprising an inactivation cleavage site was deemed
to possess enough activity at the inactivation cleavage site if it
exhibited a safety margin value of about 15 or more.
TABLE-US-00009 TABLE 9 Animal-based Assay Analysis Safety Protease
Single-Dose Full-Dosing Lethality Margin Cleavage DAS DAS Assay
LD.sub.50/DAS Site Region Modification EC.sub.50 Relative EC.sub.50
Relative LD.sub.50 EC.sub.50 Thrombin 871-895 I873insLVPGRS 1.08
30.5 ND ND ND ND Thrombin 871-895 L881insVPRGS 0.37 7.38 ND ND ND
ND Thrombin 871-895 E884insLVPRGS 0.16 25.3 0.15 46.7 1.90 12.5
Thrombin 871-895 L891insVPRG 0.12 23.3 0.19 36.8 2.74 14.8 Thrombin
.times. 2 871-895 L881insVPRGS 0.25 11.0 0.15 34.5 4.20 26.9
L891insVPRG Factor Xa 871-895 I873insEGR 0.11 46.3 0.10 70.0 2.39
23.0 Factor Xa 871-895 I873insEGR 0.09 37.2 0.26 15.3 6.69 26.9
Thrombin E884insLVPRG MMP-2 871-895 delK871NIINTSI- 0.33 10.0 ND ND
ND ND insGPLGMLSQ MMP-2 871-895 delS885NHLIDLS- 0.10 34.5 ND ND ND
ND insGPLGMLSQ MMP-9 871-895 delK871NIINTSI- 0.11 29.1 0.16 27.7
5.04 23.9 insGPLGLWAQ MMP-9 871-895 delS885NHLIDLS- 0.08 40.8 ND ND
ND ND insGPLGLWAQ u-PA 871-895 S885insPGSGKSA 0.03 36.6 ND ND ND ND
u-PA 871-895 delN886HLIDL- 0.35 3.52 ND ND ND ND insPGSGKSA t-PA
871-895 delN872IINTSI- 0.04 30.0 ND ND ND ND insPQRGRSA t-PA
871-895 S885insPQRGRSA 0.12 10.1 ND ND ND ND t-PA 871-895
delS885NHLIDL- 0.08 16.0 0.27 25.9 4.46 17.2 insPQRGRSA Furin
871-895 I870insRKKR 0.80 2.68 ND ND ND ND Furin .times. 2 871-895
delK871NII-insRKKR 0.24 8.93 ND ND ND ND delY883ES-insKKR Furin
.times. 2 871-895 delK871NII-insRKKR 0.34 6.25 ND ND ND ND S892RKK
Tryptase 871-895 K871insIVGGE 0.14 37.3 ND ND ND ND Tryptase
871-895 K871insRIVGGE 0.21 10.4 ND ND ND ND Tryptase 871-895
delN886HLIDL- 0.13 17.2 ND ND ND ND insRIVGGE ECE-1 871-895
E884insKAFA 0.05 43.1 ND ND ND ND Cathepsin L 871-895 K871insLR
0.10 34.3 ND ND ND ND Cathepsin L 871-895 K871insFR 0.27 13.0 ND ND
ND ND Control -- WT 0.05 57.0 0.07 32.4 0.88 14.2 ND is not
determined.
[0504] After the DAS analysis, a Clostridial toxin or Clostridial
toxin chimeric comprising an inactivation cleavage site was
evaluated using a mouse lethality assay in order to determine the
safety margin by comparing the ED.sub.50 with the LD.sub.50.
[0505] Although aspects of the present specification have been
described with reference to the disclosed embodiments, one skilled
in the art will readily appreciate that the specific examples
disclosed are only illustrative of these aspects and in no way
limit the present specification. Various modifications can be made
without departing from the spirit of the present specification.
Sequence CWU 1
1
53711296PRTClostridium botulinum A1 1Met Pro Phe Val Asn Lys Gln
Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15 Val Asp Ile Ala Tyr
Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30 Val Lys Ala
Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp
Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55
60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser
Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys
Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu
Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser
Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg Ser Glu Glu Leu
Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160 Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180
185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu
Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu
Ala His Glu 210 215 220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile
Ala Ile Asn Pro Asn225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu
Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 Phe Ile Asp Ser
Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe
Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305
310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile
Tyr Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn
Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val Phe Lys Ile
Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 Thr Ile Tyr Asp Gly Phe
Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400 Phe Asn Gly
Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415 Lys
Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425
430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys
435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu
Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn
Lys Gly Glu Glu465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala
Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr
Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu Asn Ile Ser Ile
Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525 Glu Leu Met
Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 Leu
Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550
555 560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala
Leu 565 570 575 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp
Tyr Val Lys 580 585 590 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe
Leu Gly Trp Val Glu 595 600 605 Gln Leu Val Tyr Asp Phe Thr Asp Glu
Thr Ser Glu Val Ser Thr Thr 610 615 620 Asp Lys Ile Ala Asp Ile Thr
Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640 Leu Asn Ile Gly
Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655 Ile Phe
Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670
Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675
680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn
Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp
Leu Ala Lys705 710 715 720 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys
Lys Met Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys
Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr Glu Glu Glu
Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 Leu Ser Ser Lys
Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780 Asn Lys
Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795
800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys
805 810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu
Ile Gly 820 825 830 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr
Leu Ser Thr Asp 835 840 845 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp
Asn Gln Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn
Ile Ile Asn Thr Ser Ile Leu Asn865 870 875 880 Leu Arg Tyr Glu Ser
Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895 Lys Ile Asn
Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910 Gln
Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920
925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser
930 935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu
Asn Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn
Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp
Thr Leu Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln Arg Val Val Phe
Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005 Asp Tyr Ile Asn
Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020 Asn
Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025
1030 1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile
Met Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp Thr His Arg Tyr
Ile Trp Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe Asp Lys Glu Leu
Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 Asp Asn Gln Ser
Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 Leu
Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn1105
1110 1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr
Met Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly Ser Val Met Thr Thr
Asn Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr Arg Gly Thr Lys
Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn Lys Asp Asn
Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180 Val
Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185
1190 1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp
Val Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys
Asn Asp Gln Gly Ile Thr 1220 1225 1230 Asn Lys Cys Lys Met Asn Leu
Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe
His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 Asn
Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys1265
1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu
Arg Pro Leu 1285 1290 1295 21296PRTClostridium botulinum A2 2Met
Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10
15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro
20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro
Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn
Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp
Ser Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu
Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp
Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro
Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile
Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145
150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Asp Val Leu Asn
Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe
Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val
Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp
Pro Ala Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala Glu His
Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240 Arg Val Phe
Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu
Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265
270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn
275 280 285 Lys Phe Lys Asp Val Ala Ser Thr Leu Asn Lys Ala Lys Ser
Ile Ile 290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val
Phe Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly
Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys
Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val Asn Phe
Phe Lys Val Ile Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys
Ala Val Phe Arg Ile Asn Ile Val Pro Asp Glu Asn Tyr 370 375 380 Thr
Ile Lys Asp Gly Phe Asn Leu Lys Gly Ala Asn Leu Ser Thr Asn385 390
395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Ser Arg Asn Phe Thr Arg
Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu
Cys Val Arg 420 425 430 Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp
Glu Gly Tyr Asn Lys 435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val
Asn Asn Trp Asp Leu Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe
Thr Asn Asp Leu Asp Lys Val Glu Glu465 470 475 480 Ile Thr Ala Asp
Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu
Ile Gln Gln Tyr Tyr Leu Thr Phe Asp Phe Asp Asn Glu Pro 500 505 510
Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515
520 525 Glu Pro Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr
Glu 530 535 540 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln
Glu Phe Glu545 550 555 560 His Gly Asp Ser Arg Ile Ile Leu Thr Asn
Ser Ala Glu Glu Ala Leu 565 570 575 Leu Lys Pro Asn Val Ala Tyr Thr
Phe Phe Ser Ser Lys Tyr Val Lys 580 585 590 Lys Ile Asn Lys Ala Val
Glu Ala Phe Met Phe Leu Asn Trp Ala Glu 595 600 605 Glu Leu Val Tyr
Asp Phe Thr Asp Glu Thr Asn Glu Val Thr Thr Met 610 615 620 Asp Lys
Ile Ala Asp Ile Thr Ile Ile Val Pro Tyr Ile Gly Pro Ala625 630 635
640 Leu Asn Ile Gly Asn Met Leu Ser Lys Gly Glu Phe Val Glu Ala Ile
645 650 655 Ile Phe Thr Gly Val Val Ala Met Leu Glu Phe Ile Pro Glu
Tyr Ala 660 665 670 Leu Pro Val Phe Gly Thr Phe Ala Ile Val Ser Tyr
Ile Ala Asn Lys 675 680 685 Val Leu Thr Val Gln Thr Ile Asn Asn Ala
Leu Ser Lys Arg Asn Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr
Thr Val Thr Asn Trp Leu Ala Lys705 710 715 720 Val Asn Thr Gln Ile
Asp Leu Ile Arg Glu Lys Met Lys Lys Ala Leu 725 730 735 Glu Asn Gln
Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln
Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760
765 Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Ser Ala Met Ile Asn Ile
770 775 780 Asn Lys Phe Leu Asp Gln Cys Ser Val Ser Tyr Leu Met Asn
Ser Met785 790 795 800 Ile Pro Tyr Ala Val Lys Arg Leu Lys Asp Phe
Asp Ala Ser Val Arg 805 810 815 Asp Val Leu Leu Lys Tyr Ile Tyr Asp
Asn Arg Gly Thr Leu Val Leu 820 825 830 Gln Val Asp Arg Leu Lys Asp
Glu Val Asn Asn Thr Leu Ser Ala Asp 835 840 845 Ile Pro Phe Gln Leu
Ser Lys Tyr Val Asp Asn Lys Lys Leu Leu Ser 850 855 860 Thr Phe Thr
Glu Tyr Ile Lys Asn Ile Val Asn Thr Ser Ile Leu Ser865 870 875 880
Ile Val Tyr Lys Lys Asp Asp Leu Ile Asp Leu Ser Arg Tyr Gly Ala 885
890 895 Lys Ile Asn Ile Gly Asp Arg Val Tyr Tyr Asp Ser Ile Asp Lys
Asn 900 905 910 Gln Ile Lys Leu Ile Asn Leu Glu Ser Ser Thr Ile Glu
Val Ile Leu 915 920 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu
Asn Phe Ser Thr Ser 930 935 940 Phe Trp Ile Lys Ile Pro Lys Tyr Phe
Ser Lys Ile Asn Leu Asn Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn
Cys Ile Glu Asn Asn Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr
Gly Glu Ile Ile Trp Thr Leu Gln Asp Asn Lys Gln 980 985 990 Asn Ile
Gln Arg Val Val Phe Lys Tyr Ser Gln Met Val Asn Ile Ser 995 1000
1005 Asp
Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010
1015 1020 Thr Lys Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln
Lys Pro1025 1030 1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser
Asn Lys Ile Met Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp Pro
Arg Arg Tyr Ile Met Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe Asp
Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 Asp
Ser Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asn Tyr 1090
1095 1100 Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Phe Asp
Pro Asn1105 1110 1115 1120Lys Tyr Val Asp Val Asn Asn Ile Gly Ile
Arg Gly Tyr Met Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly Ser Val
Val Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150 Thr Leu Tyr Glu
Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn
Glu Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170
1175 1180 Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser
Gln Ala1185 1190 1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu
Ile Pro Asp Val Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val Met
Lys Ser Lys Asp Asp Gln Gly Ile Arg 1220 1225 1230 Asn Lys Cys Lys
Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 Phe
Ile Gly Phe His Leu Tyr Asp Asn Ile Ala Lys Leu Val Ala Ser 1250
1255 1260 Asn Trp Tyr Asn Arg Gln Val Gly Lys Ala Ser Arg Thr Phe
Gly Cys1265 1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly
Trp Gly Glu Ser Ser Leu 1285 1290 1295 31292PRTClostridium
botulinum A3 3Met Pro Phe Val Asn Lys Pro Phe Asn Tyr Arg Asp Pro
Gly Asn Gly1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His Glu Gly
Val Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu
Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro
Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu
Lys Asp Asn Tyr Leu Lys Gly Val Ile Lys Leu Phe Asp 85 90 95 Arg
Ile Tyr Ser Thr Gly Leu Gly Arg Met Leu Leu Ser Phe Ile Val 100 105
110 Lys Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys
115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Glu Pro Gly Gly
Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Thr Gly Pro
Ser Ala Asp Ile145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Asp Val Phe Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr
Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu
Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly
Thr Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 Leu
Ile His Ala Ala His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230
235 240 Arg Val Leu Lys Val Lys Thr Asn Ala Tyr Tyr Glu Met Ser Gly
Leu 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly Asn
Asp Thr Asn 260 265 270 Phe Ile Asp Ser Leu Trp Gln Lys Lys Phe Ser
Arg Asp Ala Tyr Asp 275 280 285 Asn Leu Gln Asn Ile Ala Arg Ile Leu
Asn Glu Ala Lys Thr Ile Val 290 295 300 Gly Thr Thr Thr Pro Leu Gln
Tyr Met Lys Asn Ile Phe Ile Arg Lys305 310 315 320 Tyr Phe Leu Ser
Glu Asp Ala Ser Gly Lys Ile Ser Val Asn Lys Ala 325 330 335 Ala Phe
Lys Glu Phe Tyr Arg Val Leu Thr Arg Gly Phe Thr Glu Leu 340 345 350
Glu Phe Val Asn Pro Phe Lys Val Ile Asn Arg Lys Thr Tyr Leu Asn 355
360 365 Phe Asp Lys Ala Val Phe Arg Ile Asn Ile Val Pro Asp Glu Asn
Tyr 370 375 380 Thr Ile Asn Glu Gly Phe Asn Leu Glu Gly Ala Asn Ser
Asn Gly Gln385 390 395 400 Asn Thr Glu Ile Asn Ser Arg Asn Phe Thr
Arg Leu Lys Asn Phe Thr 405 410 415 Gly Leu Phe Glu Phe Tyr Lys Leu
Leu Cys Val Arg Gly Ile Ile Pro 420 425 430 Phe Lys Thr Lys Ser Leu
Asp Glu Gly Tyr Asn Lys Ala Leu Asn Tyr 435 440 445 Leu Cys Ile Lys
Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu 450 455 460 Asp Asn
Phe Thr Asn Asp Leu Asp Lys Val Glu Glu Ile Thr Ala Asp465 470 475
480 Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Ser Asp Leu Ile Gln
485 490 495 Gln Tyr Tyr Leu Thr Phe Asp Phe Asp Asn Glu Pro Glu Asn
Ile Ser 500 505 510 Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu
Glu Pro Met Pro 515 520 525 Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys
Tyr Glu Leu Asp Lys Tyr 530 535 540 Thr Met Phe His Tyr Leu Arg Ala
Gln Glu Phe Glu His Gly Asp Ser545 550 555 560 Arg Ile Ile Leu Thr
Asn Ser Ala Glu Glu Ala Leu Leu Lys Pro Asn 565 570 575 Val Ala Tyr
Thr Phe Phe Ser Ser Lys Tyr Val Lys Lys Ile Asn Lys 580 585 590 Ala
Val Glu Ala Val Ile Phe Leu Ser Trp Ala Glu Glu Leu Val Tyr 595 600
605 Asp Phe Thr Asp Glu Thr Asn Glu Val Thr Thr Met Asp Lys Ile Ala
610 615 620 Asp Ile Thr Ile Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn
Ile Gly625 630 635 640 Asn Met Val Ser Lys Gly Glu Phe Val Glu Ala
Ile Leu Phe Thr Gly 645 650 655 Val Val Ala Leu Leu Glu Phe Ile Pro
Glu Tyr Ser Leu Pro Val Phe 660 665 670 Gly Thr Phe Ala Ile Val Ser
Tyr Ile Ala Asn Lys Val Leu Thr Val 675 680 685 Gln Thr Ile Asn Asn
Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu 690 695 700 Val Tyr Lys
Tyr Thr Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln705 710 715 720
Ile Asp Leu Ile Arg Glu Lys Met Lys Lys Ala Leu Glu Asn Gln Ala 725
730 735 Glu Ala Thr Arg Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr
Glu 740 745 750 Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu
Ser Ser Lys 755 760 765 Leu Asn Arg Ser Ile Asn Arg Ala Met Ile Asn
Ile Asn Lys Phe Leu 770 775 780 Asp Gln Cys Ser Val Ser Tyr Leu Met
Asn Ser Met Ile Pro Tyr Ala785 790 795 800 Val Lys Arg Leu Lys Asp
Phe Asp Ala Ser Val Arg Asp Val Leu Leu 805 810 815 Lys Tyr Ile Tyr
Asp Asn Arg Gly Thr Leu Ile Leu Gln Val Asp Arg 820 825 830 Leu Lys
Asp Glu Val Asn Asn Thr Leu Ser Ala Asp Ile Pro Phe Gln 835 840 845
Leu Ser Lys Tyr Val Asn Asp Lys Lys Leu Leu Ser Thr Phe Thr Glu 850
855 860 Tyr Ile Lys Asn Ile Val Asn Thr Ser Ile Leu Ser Ile Val Tyr
Lys865 870 875 880 Lys Asp Asp Leu Ile Asp Leu Ser Arg Tyr Gly Ala
Lys Ile Asn Ile 885 890 895 Gly Asp Arg Val Tyr Tyr Asp Ser Ile Asp
Lys Asn Gln Ile Lys Leu 900 905 910 Ile Asn Leu Glu Ser Ser Thr Ile
Glu Val Ile Leu Lys Asn Ala Ile 915 920 925 Val Tyr Asn Ser Met Tyr
Glu Asn Phe Ser Thr Ser Phe Trp Ile Lys 930 935 940 Ile Pro Lys Tyr
Phe Ser Lys Ile Asn Leu Asn Asn Glu Tyr Thr Ile945 950 955 960 Ile
Asn Cys Ile Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr 965 970
975 Gly Glu Ile Ile Trp Thr Leu Gln Asp Asn Lys Gln Asn Ile Gln Arg
980 985 990 Val Val Phe Lys Tyr Ser Gln Met Val Asn Ile Ser Asp Tyr
Ile Asn 995 1000 1005 Arg Trp Met Phe Val Thr Ile Thr Asn Asn Arg
Leu Thr Lys Ser Lys 1010 1015 1020 Ile Tyr Ile Asn Gly Arg Leu Ile
Asp Gln Lys Pro Ile Ser Asn Leu1025 1030 1035 1040Gly Asn Ile His
Ala Ser Asn Lys Ile Met Phe Lys Leu Asp Gly Cys 1045 1050 1055 Arg
Asp Pro Arg Arg Tyr Ile Met Ile Lys Tyr Phe Asn Leu Phe Asp 1060
1065 1070 Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Ser
Gln Ser 1075 1080 1085 Asn Pro Gly Ile Leu Lys Asp Phe Trp Gly Asn
Tyr Leu Gln Tyr Asp 1090 1095 1100 Lys Pro Tyr Tyr Met Leu Asn Leu
Phe Asp Pro Asn Lys Tyr Val Asp1105 1110 1115 1120Val Asn Asn Ile
Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg 1125 1130 1135 Gly
Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Thr Leu Tyr Met 1140
1145 1150 Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Glu
Asp Asn 1155 1160 1165 Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn
Val Val Val Lys Asn 1170 1175 1180 Lys Glu Tyr Arg Leu Ala Thr Asn
Ala Ser Gln Ala Gly Val Glu Lys1185 1190 1195 1200Ile Leu Ser Ala
Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln Val 1205 1210 1215 Val
Val Met Lys Ser Lys Asp Asp Gln Gly Ile Arg Asn Lys Cys Lys 1220
1225 1230 Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Val
Gly Phe 1235 1240 1245 His Leu Tyr Asp Asn Ile Ala Lys Leu Val Ala
Ser Asn Trp Tyr Asn 1250 1255 1260 Arg Gln Val Gly Lys Ala Ser Arg
Thr Phe Gly Cys Ser Trp Glu Phe1265 1270 1275 1280Ile Pro Val Asp
Asp Gly Trp Gly Glu Ser Ser Leu 1285 1290 41296PRTClostridium
botulinum A4 4Met Pro Leu Val Asn Gln Gln Ile Asn Tyr Tyr Asp Pro
Val Asn Gly1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Lys Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys
Val Trp Val Ile Pro Glu Arg 35 40 45 Asp Ile Phe Thr Asn Pro Glu
Glu Val Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro
Ile Ser Tyr Tyr Asp Ser Ala Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu
Lys Asp Asn Tyr Leu Lys Gly Val Ile Lys Leu Phe Glu 85 90 95 Arg
Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Ile Ser Ile Val 100 105
110 Arg Gly Ile Pro Phe Trp Gly Gly Gly Lys Ile Asp Thr Glu Leu Lys
115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Ile Ile Gln Leu Asp Asp
Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn Leu Ala Ile Ile Gly Pro
Ser Ala Asn Ile145 150 155 160 Ile Glu Ser Gln Cys Ser Ser Phe Arg
Asp Asp Val Leu Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr
Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 Thr Val Gly Phe Glu
Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly
Lys Phe Ala Gln Asp Pro Ala Val Ala Leu Ala His Glu 210 215 220 Leu
Ile His Ala Glu His Arg Leu Tyr Gly Ile Ala Ile Asn Thr Asn225 230
235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ala Gly
Leu 245 250 255 Glu Val Ser Leu Glu Glu Leu Ile Thr Phe Gly Gly Asn
Asp Ala Lys 260 265 270 Phe Ile Asp Ser Leu Gln Lys Lys Glu Phe Ser
Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu
Asn Lys Ala Lys Ser Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln
Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser
Glu Asp Ala Thr Gly Lys Phe Leu Val Asp Arg Leu 325 330 335 Lys Phe
Asp Glu Leu Tyr Lys Leu Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350
Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355
360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Asp Val Asn
Tyr 370 375 380 Thr Ile His Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu
Ala Ala Asn385 390 395 400 Phe Asn Gly Gln Asn Ile Glu Ile Asn Asn
Lys Asn Phe Asp Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys
Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys 435 440 445 Ala Leu Asn Glu
Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460 Ser Pro
Ser Glu Asp Asn Phe Thr Asn Asp Leu Asp Lys Val Glu Glu465 470 475
480 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu
485 490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu Asn Phe Asn Phe Asp Asn
Glu Pro 500 505 510 Glu Asn Thr Ser Ile Glu Asn Leu Ser Ser Asp Ile
Ile Gly Gln Leu 515 520 525 Glu Pro Met Pro Asn Ile Glu Arg Phe Pro
Asn Gly Lys Lys Tyr Glu 530 535 540 Leu Asn Lys Tyr Thr Met Phe His
Tyr Leu Arg Ala Gln Glu Phe Lys545 550 555 560 His Ser Asn Ser Arg
Ile Ile Leu Thr Asn Ser Ala Lys Glu Ala Leu 565 570 575 Leu Lys Pro
Asn Ile Val Tyr Thr Phe Phe Ser Ser Lys Tyr Ile Lys 580 585 590 Ala
Ile Asn Lys Ala Val Glu Ala Val Thr Phe Val Asn Trp Ile Glu 595 600
605 Asn Leu Val Tyr Asp Phe Thr Asp Glu Thr Asn Glu Val Ser Thr Met
610 615 620 Asp Lys Ile Ala Asp Ile Thr Ile Val Ile Pro Tyr Ile Gly
Pro Ala625 630 635 640 Leu Asn Ile Gly Asn Met Ile Tyr Lys Gly Glu
Phe Val Glu Ala Ile 645 650 655 Ile Phe Ser Gly Ala Val Ile Leu Leu
Glu Ile Val Pro Glu Ile Ala 660 665 670 Leu Pro Val Leu Gly Thr Phe
Ala Leu Val Ser Tyr Val Ser Asn Lys 675 680 685 Val Leu Thr Val Gln
Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700 Lys Trp Asp
Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Ile705 710 715 720
Val Asn Thr Gln Ile Asn Leu Ile Arg Glu Lys
Met Lys Lys Ala Leu 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala
Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr Glu Glu Glu Lys
Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 Leu Ser Ser Lys Leu
Asn Glu Ser Ile Asn Ser Ala Met Ile Asn Ile 770 775 780 Asn Lys Phe
Leu Asp Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800
Ile Pro Tyr Ala Val Lys Arg Leu Lys Asp Phe Asp Ala Ser Val Arg 805
810 815 Asp Val Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile
Gly 820 825 830 Gln Val Asn Arg Leu Lys Asp Lys Val Asn Asn Thr Leu
Ser Ala Asp 835 840 845 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn
Lys Lys Leu Leu Ser 850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile
Thr Asn Ala Ser Ile Leu Ser865 870 875 880 Ile Val Tyr Lys Asp Asp
Asp Leu Ile Asp Leu Ser Arg Tyr Gly Ala 885 890 895 Glu Ile Tyr Asn
Gly Asp Lys Val Tyr Tyr Asn Ser Ile Asp Lys Asn 900 905 910 Gln Ile
Arg Leu Ile Asn Leu Glu Ser Ser Thr Ile Glu Val Ile Leu 915 920 925
Lys Lys Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930
935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn
Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser
Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr
Phe Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln Arg Val Val Phe Lys
Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005 Asp Tyr Ile Asn Arg
Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Ile 1010 1015 1020 Thr Lys
Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025 1030
1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Lys Ile Met
Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp Pro His Arg Tyr Ile
Val Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe Asp Lys Glu Leu Ser
Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 Asp Asn Gln Ser Asn
Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 Leu Gln
Tyr Asp Lys Ser Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn1105 1110
1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met
Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Asp Asn Val Met Thr Thr Asn
Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr Met Gly Thr Lys Phe
Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn Lys Asp Asn Ile
Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180 Val Val
Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185 1190
1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val
Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys Asn
Asp Gln Gly Ile Thr 1220 1225 1230 Asn Lys Cys Lys Met Asn Leu Gln
Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe His
Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 Asn Trp
Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys1265 1270
1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Arg Glu Arg
Pro Leu 1285 1290 1295 51296PRTClostridium botulinum A5 5Met Pro
Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15
Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20
25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu
Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro
Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser
Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys
Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Glu Leu
Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp
Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg
Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150
155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Asp Val Leu Asn Leu
Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser
Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp
Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro
Ala Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala Gly His Arg
Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240 Arg Val Phe Lys
Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val
Ser Phe Glu Glu Leu Arg Thr Phe Gly Glu His Asp Ala Lys 260 265 270
Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275
280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile
Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe
Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys
Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met
Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe
Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala
Val Phe Lys Ile Asn Ile Val Pro Glu Val Asn Tyr 370 375 380 Thr Ile
Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395
400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu
405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys
Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu
Gly Tyr Asn Lys 435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn
Asn Trp Asp Leu Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr
Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480 Ile Thr Ser Asp Thr
Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile
Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu
Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520
525 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
530 535 540 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu
Phe Glu545 550 555 560 His Gly Lys Ser Arg Ile Val Leu Thr Asn Ser
Val Asn Glu Ala Leu 565 570 575 Leu Asn Pro Ser Ser Val Tyr Thr Phe
Phe Ser Ser Asp Tyr Val Arg 580 585 590 Lys Val Asn Lys Ala Thr Glu
Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605 Gln Leu Val Tyr Asp
Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620 Asp Lys Ile
Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640
Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645
650 655 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile
Ala 660 665 670 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile
Ala Asn Lys 675 680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu
Ser Lys Arg Asn Glu 690 695 700 Lys Trp Gly Glu Val Tyr Lys Tyr Ile
Val Thr Asn Trp Leu Ala Lys705 710 715 720 Val Asn Thr Gln Ile Asp
Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala
Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr
Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Gly Asp 755 760 765
Leu Ser Ser Lys Leu Asn Asp Ser Ile Asn Lys Ala Met Ile Asn Ile 770
775 780 Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser
Met785 790 795 800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp
Ala Ser Leu Lys 805 810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn
Arg Gly Thr Leu Ile Gly 820 825 830 Gln Val Asp Arg Leu Lys Asp Lys
Val Asn Asn Thr Leu Ser Thr Asp 835 840 845 Ile Pro Phe Gln Leu Ser
Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu
Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn865 870 875 880 Leu
Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890
895 Glu Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn
900 905 910 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Ile
Ile Leu 915 920 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn
Phe Ser Thr Ser 930 935 940 Phe Trp Ile Lys Ile Pro Lys Tyr Phe Ser
Lys Ile Asn Leu Asn Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys
Ile Glu Asn Asn Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly
Glu Ile Ile Trp Thr Leu Gln Asp Asn Lys Gln 980 985 990 Asn Ile Gln
Arg Val Val Phe Lys Tyr Ser Gln Met Val Ala Ile Ser 995 1000 1005
Asp Tyr Ile Asn Arg Trp Ile Phe Ile Thr Ile Thr Asn Asn Arg Leu
1010 1015 1020 Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp
Gln Lys Pro1025 1030 1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala
Ser Asn Asn Ile Met Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp
Pro Gln Arg Tyr Ile Trp Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe
Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085
Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asn Tyr
1090 1095 1100 Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr
Asp Pro Asn1105 1110 1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly
Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly Ser
Ile Val Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr
Met Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165
Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val
1170 1175 1180 Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala
Ser Gln Ala1185 1190 1195 1200Gly Val Glu Lys Ile Leu Ser Val Leu
Glu Ile Pro Asp Val Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val
Met Lys Ser Lys Asn Asp Gln Gly Ile Arg 1220 1225 1230 Asn Lys Cys
Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245
Phe Ile Gly Phe His Gln Phe Asn Asn Ile Asp Lys Leu Val Ala Ser
1250 1255 1260 Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr
Phe Gly Cys1265 1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp
Gly Trp Gly Glu Ser Pro Leu 1285 1290 1295 61291PRTClostridium
botulinum B1 6Met Ser Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro
Ile Asp Asn1 5 10 15 Asp Asn Ile Ile Met Met Glu Pro Pro Phe Ala
Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp
Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys
Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg Asp
Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65 70 75 80 Thr Asn Asp
Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 Asn
Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105
110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu
115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile
Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala
Asn Leu Ile Ile145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu
Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser
Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro
Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly
Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala
Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230
235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys
Phe 245 250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu
Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser
Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg
Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys Ile Ser
Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310 315 320 Lys Asn Lys Phe
Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr
Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350
Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355
360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile
Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly
Phe Asn Ile385 390 395 400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg
Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile
Ser Lys Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys
Ser Val Lys Ala Pro Gly Ile Cys Ile
Asp 435 440 445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn
Ser Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr
Asn Thr Gln Ser Asn465 470 475 480 Tyr Ile Glu Asn Asp Phe Pro Ile
Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu
Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val
Asp Val Pro Ala Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Ile
Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540
Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp545
550 555 560 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser
Met Asp 565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly
Leu Phe Ala Gly 580 585 590 Trp Val Lys Gln Ile Val Asn Asp Phe Val
Ile Glu Ala Asn Lys Ser 595 600 605 Asn Thr Met Asp Lys Ile Ala Asp
Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 Gly Leu Ala Leu Asn Val
Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu625 630 635 640 Asn Ala Phe
Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 Glu
Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665
670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys
675 680 685 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala
Gln Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys
Glu Gly Met Tyr705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala
Leu Glu Glu Ile Ile Lys Tyr 725 730 735 Arg Tyr Asn Ile Tyr Ser Glu
Lys Glu Lys Ser Asn Ile Asn Ile Asp 740 745 750 Phe Asn Asp Ile Asn
Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765 Asp Asn Ile
Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys
Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn785 790
795 800 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu
Tyr 805 810 815 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn
Lys Tyr Leu 820 825 830 Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr
Thr Asn Asp Thr Ile 835 840 845 Leu Ile Glu Met Phe Asn Lys Tyr Asn
Ser Glu Ile Leu Asn Asn Ile 850 855 860 Ile Leu Asn Leu Arg Tyr Lys
Asp Asn Asn Leu Ile Asp Leu Ser Gly865 870 875 880 Tyr Gly Ala Lys
Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895 Asn Gln
Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900 905 910
Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915
920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln
Asn 930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys
Asn Asn Ser945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg
Ile Ile Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser
Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990 Glu Asp Ile Ser Glu Tyr
Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Leu
Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010 1015 1020
Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile1025
1030 1035 1040Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe
Ile Trp Met 1045 1050 1055 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu
Ser Gln Ser Asn Ile Glu 1060 1065 1070 Glu Arg Tyr Lys Ile Gln Ser
Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 Gly Asn Pro Leu
Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 Asn
Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu1105
1110 1115 1120Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr
Ile Asn Tyr 1125 1130 1135 Arg Asp Leu Tyr Ile Gly Glu Lys Phe Ile
Ile Arg Arg Lys Ser Asn 1140 1145 1150 Ser Gln Ser Ile Asn Asp Asp
Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 Leu Asp Phe Phe
Asn Leu Asn Gln Glu Trp Arg Val Tyr Ile Tyr Lys 1170 1175 1180 Tyr
Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp1185
1190 1195 1200Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr
Asp Glu Gln 1205 1210 1215 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys
Lys Asp Glu Glu Ser Thr 1220 1225 1230 Asp Glu Ile Gly Leu Ile Gly
Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245 Val Phe Lys Glu
Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys
Glu Val Lys Arg Lys Pro Tyr Asn Ser Lys Leu Gly Cys Asn Trp1265
1270 1275 1280Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
71291PRTClostridium botulinum B2 7Met Pro Val Thr Ile Asn Asn Phe
Asn Tyr Asn Asp Pro Ile Asp Asn1 5 10 15 Asn Asn Ile Ile Met Met
Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala
Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr
Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60
Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65
70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys
Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu
Leu Glu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg
Arg Val Pro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val
Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg
Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile145 150 155 160 Phe Gly Pro
Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile
Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185
190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu
195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser
Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu
His Gly Leu Tyr225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile
Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Ala
Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro
Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys
Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys
Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310
315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu
Gly 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr
Lys Ser Leu 340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu
Asn Tyr Lys Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser
Leu Pro Pro Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile
Tyr Thr Ile Glu Glu Gly Phe Asn Ile385 390 395 400 Ser Asp Lys Asn
Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys
Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430
Lys Ile Gln Met Cys Lys Ser Val Arg Ala Pro Gly Ile Cys Ile Asp 435
440 445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe
Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asp Thr
Gln Ser Asn465 470 475 480 Tyr Ile Glu Asn Arg Ser Ser Ile Asp Glu
Leu Ile Leu Asp Thr Asn 485 490 495 Leu Ile Ser Lys Ile Glu Leu Pro
Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Asp Val
Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Ile Phe Thr
Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr Phe
Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp545 550 555
560 Asp Ala Leu Leu Phe Ser Lys Lys Val Tyr Ser Phe Phe Ser Met Asp
565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe
Ala Gly 580 585 590 Trp Val Lys Gln Ile Val Asp Asp Phe Val Ile Glu
Ala Asn Lys Ser 595 600 605 Ser Thr Met Asp Lys Ile Ala Asp Ile Ser
Leu Ile Val Pro Tyr Ile 610 615 620 Gly Leu Ala Leu Asn Val Gly Asn
Glu Thr Ala Lys Gly Asn Phe Glu625 630 635 640 Asn Ala Phe Glu Ile
Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 Glu Leu Leu
Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670 Asp
Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680
685 Arg Asp Glu Lys Trp Ile Asp Met Tyr Gly Leu Ile Val Ala Gln Trp
690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly
Met Tyr705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu
Glu Ile Ile Lys Tyr 725 730 735 Lys Tyr Asn Ile Tyr Ser Glu Lys Glu
Lys Ser Asn Ile Asn Ile Asp 740 745 750 Phe Asn Asp Ile Asn Ser Lys
Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765 Asp Asn Ile Asn Asn
Phe Ile Asn Glu Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys Met
Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn785 790 795 800
Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805
810 815 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asp Lys His
Leu 820 825 830 Lys Thr Ile Ile Pro Phe Asp Leu Ser Lys Tyr Thr Asn
Asn Thr Ile 835 840 845 Leu Ile Glu Ile Phe Asn Lys Tyr Asn Ser Glu
Ile Leu Asn Asn Ile 850 855 860 Ile Leu Asn Leu Arg Tyr Arg Asp Asn
Asn Leu Ile Asp Leu Ser Gly865 870 875 880 Tyr Gly Ala Asn Val Glu
Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys
Leu Thr Ser Ser Thr Asn Ser Glu Ile Arg Val Thr 900 905 910 Gln Asn
Gln Asn Ile Ile Phe Asn Ser Met Phe Leu Asp Phe Ser Val 915 920 925
Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930
935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Ile Lys Asn Asn
Ser945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile
Trp Thr Leu Thr 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe
Phe Glu Tyr Ser Ile Arg 980 985 990 Lys Asp Val Ser Glu Tyr Ile Asn
Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Ser Asp Asn
Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010 1015 1020 Asn Ile
Asp Ile Lys Asp Ile Gly Glu Val Ile Ala Asn Gly Glu Ile1025 1030
1035 1040Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile
Trp Met 1045 1050 1055 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser
Gln Ser Asn Ile Lys 1060 1065 1070 Glu Ile Tyr Lys Ile Gln Ser Tyr
Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 Gly Asn Pro Leu Met
Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 Asn Lys
Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Ser Val Gly Glu1105 1110
1115 1120Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Asn Tyr Ile
Asn Tyr 1125 1130 1135 Arg Asn Leu Tyr Ile Gly Glu Lys Phe Ile Ile
Arg Arg Lys Ser Asn 1140 1145 1150 Ser Gln Ser Ile Asn Asp Asp Ile
Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 Leu Asp Phe Phe Asn
Ser Asn Arg Glu Trp Arg Val Tyr Ala Tyr Lys 1170 1175 1180 Asp Phe
Lys Glu Glu Glu Lys Lys Leu Phe Leu Ala Asn Ile Tyr Asp1185 1190
1195 1200Ser Asn Glu Phe Tyr Lys Thr Ile Gln Ile Lys Glu Tyr Asp
Glu Gln 1205 1210 1215 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys
Asp Glu Glu Ser Thr 1220 1225 1230 Asp Glu Ile Gly Leu Ile Gly Ile
His Arg Phe Tyr Glu Ser Gly Thr 1235 1240 1245 Val Phe Lys Asn Tyr
Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys Glu
Val Lys Arg Lys Pro Tyr Asn Ser Asp Leu Gly Cys Asn Trp1265 1270
1275 1280Lys Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
81291PRTClostridium botulinum B3 8Met Pro Val Thr Ile Asn Asn Phe
Asn Tyr Asn Asp Pro Ile Asp Asn1 5 10 15 Asp Asn Ile Ile Met Met
Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala
Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr
Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60
Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65
70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys
Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu
Leu Glu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg
Arg Val Pro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val
Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg
Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile145 150 155 160 Phe Gly Pro
Gly Pro Val Leu Asn Glu Asn Glu
Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu
Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr
Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser
Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile
Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240
Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245
250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr
Phe 260 265 270 Gly Gly Gln Asp Pro Arg Ile Ile Thr Pro Ser Thr Asp
Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile
Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys Ile Ser Asp Pro
Asn Ile Asn Ile Asn Ile Tyr305 310 315 320 Lys Asn Lys Phe Lys Asp
Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser Ile
Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350 Met Phe
Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365
Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370
375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn
Ile385 390 395 400 Ser Asp Lys Asn Met Glu Lys Glu Tyr Arg Gly Gln
Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys
Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val
Arg Ala Pro Gly Ile Cys Ile Asp 435 440 445 Val Asp Asn Glu Asp Leu
Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser
Lys Asn Glu Arg Ile Glu Tyr Asp Thr Gln Ser Asn465 470 475 480 Tyr
Ile Glu Asn Arg Ser Ser Ile Asp Glu Leu Ile Leu Asp Thr Asn 485 490
495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr
500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala
Ile Lys 515 520 525 Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr
Leu Tyr Ser Gln 530 535 540 Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser
Leu Thr Ser Ser Phe Asp545 550 555 560 Asp Ala Leu Leu Phe Ser Asn
Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys Thr Ala
Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val Lys
Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Ser
Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615
620 Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe
Glu625 630 635 640 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu
Glu Phe Ile Pro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Ala Phe
Leu Leu Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys
Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 Arg Asp Glu Lys Trp Ile
Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 Leu Ser Thr Val
Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr705 710 715 720 Lys
Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730
735 Lys Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp
740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln
Ala Ile 755 760 765 Asp Asn Ile Asn Asn Phe Ile Asn Glu Cys Ser Val
Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu Ala Val Glu Lys
Leu Leu Asp Phe Asp Asn785 790 795 800 Thr Leu Lys Lys Asn Leu Leu
Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly Ser Ala
Glu Tyr Glu Lys Ser Lys Val Asp Lys His Leu 820 825 830 Lys Thr Ile
Ile Pro Phe Asp Leu Ser Met Tyr Thr Asn Asn Thr Ile 835 840 845 Leu
Ile Glu Ile Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855
860 Ile Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser
Gly865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asn Gly Val Glu
Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn
Ser Lys Ile Arg Val Thr 900 905 910 Gln Asn Gln Asp Ile Ile Phe Asn
Ser Met Phe Leu Asp Phe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile
Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930 935 940 Tyr Ile His Asn
Glu Tyr Thr Ile Ile Asn Cys Ile Lys Asn Asn Ser945 950 955 960 Gly
Trp Lys Ile Ser Ile Arg Gly Asn Lys Ile Ile Trp Thr Leu Thr 965 970
975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Ser Ile Arg
980 985 990 Lys Asp Val Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr
Ile Thr 995 1000 1005 Asn Asn Ser Asp Asn Ala Lys Ile Tyr Ile Asn
Gly Lys Leu Glu Ser 1010 1015 1020 Asn Ile Asp Ile Lys Asp Ile Gly
Glu Val Ile Ala Asn Gly Glu Ile1025 1030 1035 1040Ile Phe Lys Leu
Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile Trp Met 1045 1050 1055 Lys
Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Lys 1060
1065 1070 Glu Ile Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp
Phe Trp 1075 1080 1085 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr
Met Phe Asn Ala Gly 1090 1095 1100 Asn Lys Asn Ser Tyr Ile Lys Leu
Lys Lys Asp Ser Ser Val Gly Glu1105 1110 1115 1120Ile Leu Thr Arg
Ser Lys Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr 1125 1130 1135 Arg
Asn Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140
1145 1150 Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr
Ile Tyr 1155 1160 1165 Leu Asp Phe Phe Asn Leu Asn Gln Glu Trp Arg
Val Tyr Ala Tyr Lys 1170 1175 1180 Asp Phe Lys Lys Lys Glu Glu Lys
Leu Phe Leu Ala Asn Ile Tyr Asp1185 1190 1195 1200Ser Asn Glu Phe
Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln 1205 1210 1215 Pro
Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220
1225 1230 Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser
Gly Ile 1235 1240 1245 Val Phe Lys Asp Tyr Lys Asp Tyr Phe Cys Ile
Ser Lys Trp Tyr Leu 1250 1255 1260 Lys Glu Val Lys Arg Lys Pro Tyr
Asn Pro Asn Leu Gly Cys Asn Trp1265 1270 1275 1280Gln Phe Ile Pro
Lys Asp Glu Gly Trp Ile Glu 1285 1290 91291PRTClostridium botulinum
Bnp 9Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp
Asn1 5 10 15 Asp Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly
Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile
Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu
Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg Asp Val Cys
Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65 70 75 80 Thr Asn Asp Lys Lys
Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile
Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 Ile
Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120
125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn
130 135 140 Pro Gly Glu Val Glu Gln Lys Lys Gly Ile Phe Ala Asn Leu
Ile Ile145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu
Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu
Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr
Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser
Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile
Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240
Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245
250 255 Phe Met Gln Ser Thr Asp Thr Ile Gln Ala Glu Glu Leu Tyr Thr
Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Ser Pro Ser Thr Asp
Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile
Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys Ile Ser Asp Pro
Asn Ile Asn Ile Asn Ile Tyr305 310 315 320 Lys Asn Lys Phe Lys Asp
Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser Ile
Asp Val Glu Ser Phe Asn Lys Leu Tyr Lys Ser Leu 340 345 350 Met Phe
Gly Phe Thr Glu Ile Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365
Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370
375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn
Ile385 390 395 400 Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gln
Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys
Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val
Lys Val Pro Gly Ile Cys Ile Asp 435 440 445 Val Asp Asn Glu Asn Leu
Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser
Lys Asn Glu Arg Val Glu Tyr Asn Thr Gln Asn Asn465 470 475 480 Tyr
Ile Gly Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490
495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr
500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala
Ile Lys 515 520 525 Lys Val Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr
Leu Tyr Ser Gln 530 535 540 Thr Phe Pro Leu Asn Ile Arg Asp Ile Ser
Leu Thr Ser Ser Phe Asp545 550 555 560 Asp Ala Leu Leu Val Ser Ser
Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys Thr Ala
Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val Lys
Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Ser
Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615
620 Gly Leu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe
Glu625 630 635 640 Ser Ala Phe Glu Ile Ala Gly Ser Ser Ile Leu Leu
Glu Phe Ile Pro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Val Phe
Leu Leu Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys
Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 Arg Val Glu Lys Trp Ile
Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 Leu Ser Thr Val
Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr705 710 715 720 Lys
Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730
735 Lys Tyr Asn Ile Tyr Ser Glu Glu Glu Lys Ser Asn Ile Asn Ile Asn
740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Asp Gly Ile Asn Gln
Ala Met 755 760 765 Asp Asn Ile Asn Asp Phe Ile Asn Glu Cys Ser Val
Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu Ala Val Lys Lys
Leu Leu Asp Phe Asp Asn785 790 795 800 Thr Leu Lys Lys Asn Leu Leu
Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly Ser Val
Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu 820 825 830 Lys Thr Ile
Ile Pro Phe Asp Leu Ser Thr Tyr Thr Asn Asn Glu Ile 835 840 845 Leu
Ile Lys Ile Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855
860 Ile Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser
Gly865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys
Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asp
Ser Lys Ile Arg Val Thr 900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn
Ser Met Phe Leu Asp Phe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile
Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn 930 935 940 Tyr Ile His Asn
Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser945 950 955 960 Gly
Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970
975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg
980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr
Ile Thr 995 1000 1005 Asn Asn Leu Asp Asn Ala Lys Ile Tyr Ile Asn
Gly Thr Leu Glu Ser 1010 1015 1020 Asn Met Asp Ile Lys Asp Ile Gly
Glu Val Ile Val Asn Gly Glu Ile1025 1030 1035 1040Thr Phe Lys Leu
Asp Gly Asp Val Asp Arg Thr Gln Phe Ile Trp Met 1045 1050 1055 Lys
Tyr Phe Ser Ile Phe Asn Thr Gln Leu Asn Gln Ser Asn Ile Lys 1060
1065 1070 Glu Ile Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp
Phe Trp 1075 1080 1085 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr
Met Phe Asn Ala Gly 1090 1095 1100 Asn Lys Asn Ser Tyr Ile Lys Leu
Val Lys Asp Ser Ser Val Gly Glu1105 1110 1115 1120Ile Leu Ile Arg
Ser Lys Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr 1125 1130 1135 Arg
Asn Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140
1145 1150 Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr
Ile His 1155 1160 1165 Leu Asp Phe Val Asn Ser Asn Glu Glu Trp Arg
Val Tyr Ala Tyr Lys 1170 1175 1180
Asn Phe Lys Glu Gln Glu Gln Lys Leu Phe Leu Ser Ile Ile Tyr Asp1185
1190 1195 1200Ser Asn Glu Phe Tyr Lys Thr Ile Gln Ile Lys Glu Tyr
Asp Glu Gln 1205 1210 1215 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys
Lys Asp Glu Glu Ser Thr 1220 1225 1230 Asp Asp Ile Gly Leu Ile Gly
Ile His Arg Phe Tyr Glu Ser Gly Val 1235 1240 1245 Leu Arg Lys Lys
Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys
Glu Val Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp1265
1270 1275 1280Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
101291PRTClostridium botulinum Bbv 10Met Pro Val Thr Ile Asn Asn
Phe Asn Tyr Asn Asp Pro Ile Asp Asn1 5 10 15 Asn Asn Ile Ile Met
Met Glu Pro Pro Phe Ala Arg Gly Met Gly Arg 20 25 30 Tyr Tyr Lys
Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg
Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55
60 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu
Asn65 70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile
Lys Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys
Leu Leu Glu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp
Arg Arg Val Pro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser
Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu
Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile145 150 155 160 Phe Gly
Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175
Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180
185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln
Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe
Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val
Leu His Gly Leu Tyr225 230 235 240 Gly Ile Lys Val Asn Asp Leu Pro
Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp
Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp
Pro Ser Ile Ile Ser Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp
Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300
Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305
310 315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser
Glu Gly 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu
Tyr Lys Ser Leu 340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala
Glu Asn Tyr Lys Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp
Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu
Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile385 390 395 400 Ser Asp Lys
Asn Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn
Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425
430 Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp
435 440 445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser
Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Ile Ala Tyr Asn
Thr Gln Asn Asn465 470 475 480 Tyr Ile Glu Asn Asp Phe Ser Ile Asn
Glu Leu Ile Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu Leu
Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Tyr
Val Pro Val Tyr Lys Lys Gln Pro Ala Ile Lys 515 520 525 Lys Ile Phe
Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr
Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp545 550
555 560 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met
Asp 565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu
Phe Ala Gly 580 585 590 Trp Val Lys Gln Ile Val Asp Asp Phe Val Ile
Glu Ala Asn Lys Ser 595 600 605 Ser Thr Met Asp Lys Ile Ala Asp Ile
Ser Leu Ile Val Pro Tyr Ile 610 615 620 Gly Leu Ala Leu Asn Val Gly
Asn Glu Thr Ala Lys Gly Asn Phe Glu625 630 635 640 Asn Ala Phe Glu
Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 Glu Leu
Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670
Asp Asn Lys Asn Lys Ile Ile Glu Thr Ile Asn Ser Ala Leu Thr Lys 675
680 685 Arg Asp Glu Lys Trp Ile Asp Met Tyr Gly Leu Ile Val Ala Gln
Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu
Gly Met Tyr705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu
Glu Glu Ile Ile Lys Tyr 725 730 735 Lys Tyr Asn Ile Tyr Ser Glu Lys
Glu Arg Ser Asn Ile Asn Ile Asp 740 745 750 Phe Asn Asp Val Asn Ser
Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765 Asp Asn Ile Asn
Asn Phe Ile Asn Glu Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys
Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn785 790 795
800 Thr Leu Arg Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr
805 810 815 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asp Lys
Tyr Leu 820 825 830 Lys Thr Ser Ile Pro Phe Asp Leu Ser Thr Tyr Thr
Asn Asn Thr Ile 835 840 845 Leu Ile Glu Ile Phe Asn Lys Tyr Asn Ser
Asp Ile Leu Asn Asn Ile 850 855 860 Ile Leu Asn Leu Arg Tyr Arg Asp
Asn Lys Leu Ile Asp Leu Ser Gly865 870 875 880 Tyr Gly Ala Lys Val
Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys 885 890 895 Asn Gln Phe
Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Ile 900 905 910 Gln
Asn Gln Asn Ile Ile Phe Asn Ser Met Phe Leu Asp Phe Ser Val 915 920
925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn
930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn
Asn Ser945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Met Ile
Ile Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Ile Lys Ser Val
Phe Phe Glu Tyr Ser Ile Lys 980 985 990 Glu Asp Ile Ser Glu Tyr Ile
Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Ser Asp
Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010 1015 1020 His
Ile Asp Ile Arg Asp Ile Arg Glu Val Ile Ala Asn Asp Glu Ile1025
1030 1035 1040Ile Phe Lys Leu Asp Gly Asn Ile Asp Arg Thr Gln Phe
Ile Trp Met 1045 1050 1055 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu
Ser Gln Ser Asn Ile Glu 1060 1065 1070 Glu Ile Tyr Lys Ile Gln Ser
Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 Gly Asn Pro Leu
Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 Asn
Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Ser Val Gly Glu1105
1110 1115 1120Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr
Ile Asn Tyr 1125 1130 1135 Arg Asp Leu Tyr Ile Gly Glu Lys Phe Ile
Ile Arg Arg Lys Ser Asn 1140 1145 1150 Ser Gln Ser Ile Asn Asp Asp
Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 Leu Asp Phe Phe
Asn Leu Asn Gln Glu Trp Arg Val Tyr Met Tyr Lys 1170 1175 1180 Tyr
Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp1185
1190 1195 1200Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr
Asp Glu Gln 1205 1210 1215 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys
Lys Asp Glu Glu Ser Thr 1220 1225 1230 Asp Glu Ile Gly Leu Ile Gly
Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245 Val Phe Lys Glu
Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys
Glu Val Lys Arg Lys Pro Tyr Asn Ser Lys Leu Gly Cys Asn Trp1265
1270 1275 1280Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
111291PRTClostridium botulinum C1-1 11Met Pro Ile Thr Ile Asn Asn
Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15 Lys Asn Ile Leu Tyr
Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30 Pro Glu Lys
Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45 Arg
Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55
60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr
Asp65 70 75 80 Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu
Phe Lys Arg 85 90 95 Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile
Tyr Arg Leu Ser Thr 100 105 110 Asp Ile Pro Phe Pro Gly Asn Asn Asn
Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe Asp Val Asp Phe Asn Ser
Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 Asn Trp Val Lys Thr
Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160 Pro Arg
Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175
Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180
185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn
Asp 195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met
Asp Pro Ile 210 215 220 Leu Ile Leu Met His Glu Leu Asn His Ala Met
His Asn Leu Tyr Gly225 230 235 240 Ile Ala Ile Pro Asn Asp Gln Thr
Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 Phe Tyr Ser Gln Tyr Asn
Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270 Phe Gly Gly Pro
Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285 Phe Glu
Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300
Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305
310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val
Glu Ser 325 330 335 Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val
Glu Leu Tyr Asn 340 345 350 Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn
Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Gln Asn Arg Lys Ile Tyr Leu
Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 Ala Asn Ile Leu Asp Asp
Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400 Ile Pro Lys
Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415 Arg
Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425
430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn
435 440 445 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp
Leu Pro 450 455 460 Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile
Phe Leu Arg Lys465 470 475 480 Asp Ile Asn Glu Glu Thr Glu Val Ile
Tyr Tyr Pro Asp Asn Val Ser 485 490 495 Val Asp Gln Val Ile Leu Ser
Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510 Asp Leu Leu Tyr Pro
Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525 Glu Asn Gln
Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540 Asn
Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550
555 560 Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser
Ala 565 570 575 Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val
Asn Ala Gly 580 585 590 Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn
Asp Val Val Glu Asp 595 600 605 Phe Thr Thr Asn Ile Leu Arg Lys Asp
Thr Leu Asp Lys Ile Ser Asp 610 615 620 Val Ser Ala Ile Ile Pro Tyr
Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640 Ser Val Arg Arg
Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655 Thr Ile
Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670
Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675
680 685 Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp
Ser 690 695 700 Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile
Thr Gln Phe705 710 715 720 Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser
Leu Asn Tyr Gln Ala Gly 725 730 735 Ala Ile Lys Ala Lys Ile Asp Leu
Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 Asp Lys Glu Asn Ile Lys
Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765 Asp Val Lys Ile
Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780 Glu Cys
Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795
800 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn
805 810 815 Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp
Lys Leu 820 825 830 Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile
Pro Phe Asn Ile 835 840 845 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys
Asp Ile Ile Asn Glu Tyr 850 855 860 Phe Asn Asn Ile Asn Asp Ser Lys
Ile Leu Ser Leu Gln Asn Arg Lys865 870 875 880 Asn Thr Leu Val Asp
Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895 Gly Asp Val
Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900
905 910 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu
Asn 915 920 925 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser
Phe Trp Ile 930 935 940 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly
Tyr Thr Ile Ile Asp945 950 955 960 Ser Val Lys Asn Asn Ser Gly Trp
Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975 Leu Val Phe Thr Leu Lys
Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 980 985 990 Phe Ser Tyr Asp
Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005 Phe
Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile 1010
1015 1020 Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr
Gly Ile1025 1030 1035 1040Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile
Asn Lys Ile Pro Asp Thr 1045 1050 1055 Gly Leu Ile Thr Ser Asp Ser
Asp Asn Ile Asn Met Trp Ile Arg Asp 1060 1065 1070 Phe Tyr Ile Phe
Ala Lys Glu Leu Asp Gly Lys Asp Ile Asn Ile Leu 1075 1080 1085 Phe
Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 1090
1095 1100 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile Asp
Tyr Leu1105 1110 1115 1120Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln
Ile Val Phe Asn Thr Arg 1125 1130 1135 Arg Asn Asn Asn Asp Phe Asn
Glu Gly Tyr Lys Ile Ile Ile Lys Arg 1140 1145 1150 Ile Arg Gly Asn
Thr Asn Asp Thr Arg Val Arg Gly Gly Asp Ile Leu 1155 1160 1165 Tyr
Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys 1170
1175 1180 Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp Ile
Tyr Ala1185 1190 1195 1200Ile Gly Leu Arg Glu Gln Thr Lys Asp Ile
Asn Asp Asn Ile Ile Phe 1205 1210 1215 Gln Ile Gln Pro Met Asn Asn
Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe 1220 1225 1230 Lys Ser Asn Phe
Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245 Thr
Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 1250
1255 1260 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu
Ser Thr1265 1270 1275 1280Ser Thr His Trp Gly Phe Val Pro Val Ser
Glu 1285 1290 121280PRTClostridium botulinum C1-2 12Met Pro Ile Thr
Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15 Lys Asn
Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30
Pro Glu Lys Ala Phe Arg Ile Ile Gly Asn Ile Trp Val Ile Pro Asp 35
40 45 Arg Phe Ser Arg Asp Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg
Val 50 55 60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu
Ser Thr Asp65 70 75 80 Ser Glu Lys Asp Thr Phe Leu Lys Glu Ile Ile
Lys Leu Phe Lys Arg 85 90 95 Ile Asn Ser Arg Glu Ile Gly Glu Glu
Leu Ile Tyr Arg Leu Ala Thr 100 105 110 Asp Ile Pro Phe Pro Gly Asn
Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe Asp Val Asp Phe
Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 Asn Trp Val
Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160
Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165
170 175 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile
Ile 180 185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala
Thr Asn Asn 195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe
Cys Met Asp Pro Ile 210 215 220 Leu Ile Leu Met His Glu Leu Asn His
Thr Met His Asn Leu Tyr Gly225 230 235 240 Ile Ala Ile Pro Asn Asp
Gln Arg Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 Phe Tyr Ser Gln
Tyr Lys Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270 Phe Gly
Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Gly Arg Lys Tyr 275 280 285
Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290
295 300 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile
Gly305 310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe
Val Val Glu Ser 325 330 335 Ser Gly Glu Val Ala Val Asp Arg Asn Lys
Phe Ala Glu Leu Tyr Lys 340 345 350 Glu Leu Thr Gln Ile Phe Thr Glu
Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Gln Asn Arg Lys Ile
Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 Ala Asn Ile Leu
Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400 Ile
Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410
415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu
420 425 430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu
Tyr Asn 435 440 445 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn
Thr Asp Leu Pro 450 455 460 Phe Ile Gly Asp Ile Ser Asp Ile Lys Thr
Asp Ile Phe Leu Ser Lys465 470 475 480 Asp Ile Asn Val Glu Thr Glu
Val Ile Asp Tyr Pro Asp Asn Val Ser 485 490 495 Val Asp Gln Val Ile
Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510 Asp Leu Leu
Tyr Pro Ile Ile Glu Gly Glu Ser Gln Val Leu Pro Gly 515 520 525 Glu
Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535
540 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val
Glu545 550 555 560 Asp Phe Thr Phe Thr Thr Ser Ile Glu Glu Ala Leu
Asp Asn Ser Gly 565 570 575 Lys Val Tyr Thr Tyr Phe Pro Lys Leu Ala
Asp Lys Val Asn Thr Gly 580 585 590 Val Gln Gly Gly Leu Phe Leu Met
Trp Ala Asn Asp Val Val Glu Asp 595 600 605 Phe Thr Thr Asn Ile Leu
Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620 Val Ser Ala Ile
Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640 Ser
Val Arg Arg Glu Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650
655 Thr Ile Leu Leu Glu Ala Phe Gln Glu Phe Thr Ile Pro Ala Leu Gly
660 665 670 Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile
Ile Lys 675 680 685 Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg
Trp Lys Asp Ser 690 695 700 Tyr Glu Trp Met Ile Gly Thr Trp Leu Ser
Arg Ile Thr Thr Gln Phe705 710 715 720 Asn Asn Ile Ser Tyr Gln Met
Tyr Asp Ser Leu Asn Tyr Gln Ala Asp 725 730 735 Ala Ile Lys Asp Lys
Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 Asp Lys Glu
Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765 Asp
Ile Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775
780 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val
Ile785 790 795 800 Asp Glu Leu Asn Lys Phe Asp Leu Lys Thr Lys Thr
Glu Leu Ile Asn 805 810 815 Leu Ile Asp Ser His Asn Ile Ile Leu Val
Gly Glu Val Asp Arg Leu 820 825 830 Lys Ala Lys Val Asn Glu Ser Phe
Glu Asn Thr Ile Pro Phe Asn Ile 835 840 845 Phe Ser Tyr Thr Asn Asn
Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860 Phe Asn Ser Ile
Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys865 870 875 880 Asn
Ala Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Leu Glu 885 890
895 Gly Asp Val Gln Val Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser
900 905 910 Ser Ser Gly Asp Lys Ile Ile Val Asn Leu Asn Asn Asn Ile
Leu Tyr 915 920 925 Ser Ala Ile Tyr Glu Asn Ser Ser Val Ser Phe Trp
Ile Lys Ile Ser 930 935 940 Lys Asp Leu Thr Asn Ser His Asn Glu Tyr
Thr Ile Ile Asn Ser Ile945 950 955 960 Lys Gln Asn Ser Gly Trp Lys
Leu Cys Ile Arg Asn Gly Asn Ile Glu 965 970 975 Trp Ile Leu Gln Asp
Ile Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp 980 985 990 Tyr Ser Glu
Ser Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe 995 1000 1005
Val Thr Ile Thr Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn
1010 1015 1020 Gly Glu Leu Lys Gln Ser Glu Arg Ile Glu Asp Leu Asn
Glu Val Lys1025 1030 1035 1040Leu Asp Lys Thr Ile Val Phe Gly Ile
Asp Glu Asn Ile Asp Glu Asn 1045 1050 1055 Gln Met Leu Trp Ile Arg
Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser 1060 1065 1070 Asn Glu Asp
Ile Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn Val 1075 1080 1085
Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr
1090 1095 1100 Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro
Lys Ser Asn1105 1110 1115 1120Ile Leu Val Leu Val Gln Tyr Pro Asp
Arg Ser Lys Leu Tyr Thr Gly 1125 1130 1135 Asn Pro Ile Thr Ile Lys
Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg 1140 1145 1150 Ile Leu Asn
Gly Asp Asn Ile Met Phe His Met Leu Tyr Asn Ser Gly 1155 1160 1165
Lys Tyr Met Ile Ile Arg Asp Thr Asp Thr Ile Tyr Ala Ile Glu Gly
1170 1175 1180 Arg Glu Cys Ser Lys Asn Cys Val Tyr Ala Leu Lys Leu
Gln Ser Asn1185 1190 1195 1200Leu Gly Asn Tyr Gly Ile Gly Ile Phe
Ser Ile Lys Asn Ile Val Ser 1205 1210 1215 Gln Asn Lys Tyr Cys Ser
Gln Ile Phe Ser Ser Phe Met Lys Asn Thr 1220 1225 1230 Met Leu Leu
Ala Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Glu Asn 1235 1240 1245
Ala Tyr Thr Pro Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser
1250 1255 1260 Thr Ser Ser Phe Trp Lys Phe Ile Ser Arg Asp Pro Gly
Trp Val Glu1265 1270 1275 1280131276PRTClostridium botulinum D1
13Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1
5 10 15 Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr
Thr 20 25 30 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val
Ile Pro Glu 35 40 45 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser
Lys Pro Pro Arg Pro 50 55 60 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp
Pro Ser Tyr Leu Ser Thr Asp65 70 75 80 Glu Gln Lys Asp Thr Phe Leu
Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Glu Arg Asp
Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110 Gly Ser Pro
Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125 Phe
Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135
140 Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe
Gly145 150 155 160 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu
Thr Leu Gln Gly 165 170 175 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe
Gly Thr Leu Ser Ile Leu 180 185 190 Lys Val Ala Pro Glu Phe Leu Leu
Thr Phe Ser Asp Val Thr Ser Asn 195 200 205 Gln Ser Ser Ala Val Leu
Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220 Ile Ala Leu Met
His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly225 230 235 240 Ile
Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250
255 Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr
260 265 270 Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg
Ser Gln 275 280 285 Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile
Ala Lys Arg Leu 290 295 300 Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser
Trp Ile Ser Asn Ile Asp305 310 315 320 Lys Tyr Lys Lys Ile Phe Ser
Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335 Thr Gly Asn Phe Val
Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350 Asp Leu Thr
Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365 Val
Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375
380 Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe
Asn385 390 395 400 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly
Gln Asn Ile Glu 405 410 415 Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser
Glu Ser Val Val Asp Leu 420 425 430 Phe Thr Lys Val Cys Leu Arg Leu
Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445 Thr Cys Ile Lys Val Lys
Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460 Asp Ser Ile Ser
Gln Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu465 470 475 480 Thr
Asn Val Gln Asn Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile 485 490
495 Leu Asp Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu
500 505 510 Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu
Ile Val 515 520 525 Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu
Asn Ser Tyr Tyr 530 535 540 Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn
Val Glu Asn Ile Thr Leu545 550 555 560 Thr Thr Ser Val Glu Glu Ala
Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575 Phe Leu Pro Ser Leu
Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly 580 585 590 Leu Phe Leu
Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 Ile
Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615
620 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu
Arg625 630 635 640 Gly Asn Phe Asn Gln
Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655 Glu Gly Phe
Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670 Tyr
Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680
685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met
690 695 700 Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His
Ile Asn705 710 715 720 Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala
Asp Ala Ile Lys Ala 725 730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr
Ser Gly Ser Asp Lys Glu Asn 740 745 750 Ile Lys Ser Gln Val Glu Asn
Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765 Ser Glu Ala Met Asn
Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780 Thr Tyr Leu
Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn785 790 795 800
Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805
810 815 His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys
Val 820 825 830 Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe
Ser Tyr Thr 835 840 845 Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu
Tyr Phe Asn Ser Ile 850 855 860 Asn Asp Ser Lys Ile Leu Ser Leu Gln
Asn Lys Lys Asn Ala Leu Val865 870 875 880 Asp Thr Ser Gly Tyr Asn
Ala Glu Val Arg Val Gly Asp Asn Val Gln 885 890 895 Leu Asn Thr Ile
Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910 Lys Ile
Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925
Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930
935 940 Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn
Ser945 950 955 960 Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu
Trp Ile Leu Gln 965 970 975 Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile
Phe Asp Tyr Ser Glu Ser 980 985 990 Leu Ser His Thr Gly Tyr Thr Asn
Lys Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Ile Met Gly
Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu Lys 1010 1015 1020 Gln Ser
Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr1025 1030
1035 1040Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gln Met
Leu Trp 1045 1050 1055 Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu
Ser Asn Glu Asp Ile 1060 1065 1070 Asn Ile Val Tyr Glu Gly Gln Ile
Leu Arg Asn Val Ile Lys Asp Tyr 1075 1080 1085 Trp Gly Asn Pro Leu
Lys Phe Asp Thr Glu Tyr Tyr Ile Ile Asn Asp 1090 1095 1100 Asn Tyr
Ile Asp Arg Tyr Ile Ala Pro Glu Ser Asn Val Leu Val Leu1105 1110
1115 1120Val Gln Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro
Ile Thr 1125 1130 1135 Ile Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser
Arg Ile Leu Asn Gly 1140 1145 1150 Asp Asn Ile Ile Leu His Met Leu
Tyr Asn Ser Arg Lys Tyr Met Ile 1155 1160 1165 Ile Arg Asp Thr Asp
Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser 1170 1175 1180 Gln Asn
Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr1185 1190
1195 1200Gly Ile Gly Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn
Lys Tyr 1205 1210 1215 Cys Ser Gln Ile Phe Ser Ser Phe Arg Glu Asn
Thr Met Leu Leu Ala 1220 1225 1230 Asp Ile Tyr Lys Pro Trp Arg Phe
Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245 Val Ala Val Thr Asn
Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe 1250 1255 1260 Trp Lys
Phe Ile Ser Arg Asp Pro Gly Trp Val Glu1265 1270 1275
141285PRTClostridium botulinum D2 14Met Thr Trp Pro Val Lys Asp Phe
Asn Tyr Ser Asp Pro Val Asn Asp1 5 10 15 Asn Asp Ile Leu Tyr Leu
Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30 Pro Val Lys Ala
Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45 Arg Phe
Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60
Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp65
70 75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe
Lys Arg 85 90 95 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn
Tyr Leu Val Val 100 105 110 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr
Pro Glu Asp Thr Phe Asp 115 120 125 Phe Thr Arg His Thr Thr Asn Ile
Ala Val Glu Lys Phe Glu Asn Gly 130 135 140 Ser Trp Lys Val Thr Asn
Ile Ile Thr Pro Ser Val Leu Ile Phe Gly145 150 155 160 Pro Leu Pro
Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175 Gln
Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185
190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn
195 200 205 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp
Pro Val 210 215 220 Ile Ala Leu Met His Glu Leu Thr His Ser Leu His
Gln Leu Tyr Gly225 230 235 240 Ile Asn Ile Pro Ser Asp Lys Arg Ile
Arg Pro Gln Val Ser Glu Gly 245 250 255 Phe Phe Ser Gln Asp Gly Pro
Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270 Phe Gly Gly Ser Asp
Val Glu Ile Ile Pro Gln Ile Glu Arg Leu Gln 275 280 285 Leu Arg Glu
Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300 Asn
Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ser Ser Asn Ile Asp305 310
315 320 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp
Asn 325 330 335 Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser
Leu Tyr Ser 340 345 350 Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr
Ser Ser Gln Tyr Asn 355 360 365 Val Lys Asn Arg Thr His Tyr Phe Ser
Lys His Tyr Leu Pro Val Phe 370 375 380 Ala Asn Ile Leu Asp Asp Asn
Ile Tyr Thr Ile Ile Asn Gly Phe Asn385 390 395 400 Leu Thr Thr Lys
Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415 Arg Asn
Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430
Phe Thr Lys Val Cys Leu Arg Leu Thr Arg Asn Ser Arg Asp Asp Ser 435
440 445 Thr Cys Ile Gln Val Lys Asn Asn Thr Leu Pro Tyr Val Ala Asp
Lys 450 455 460 Asp Ser Ile Ser Gln Glu Ile Phe Glu Ser Gln Ile Ile
Thr Asp Glu465 470 475 480 Thr Asn Val Glu Asn Tyr Ser Asp Asn Phe
Ser Leu Asp Glu Ser Ile 485 490 495 Leu Asp Ala Lys Val Pro Thr Asn
Pro Glu Ala Val Asp Pro Leu Leu 500 505 510 Pro Asn Val Asn Met Glu
Pro Leu Asn Val Pro Gly Glu Glu Glu Val 515 520 525 Phe Tyr Asp Asp
Ile Thr Lys Asp Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540 Tyr Leu
Glu Ala Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu545 550 555
560 Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr
565 570 575 Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln
Ala Gly 580 585 590 Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp
Phe Thr Thr Asn 595 600 605 Ile Met Lys Lys Asp Thr Leu Asp Lys Ile
Ser Asp Val Ser Ala Ile 610 615 620 Ile Pro Tyr Ile Gly Pro Ala Leu
Asn Ile Gly Asn Ser Ala Leu Arg625 630 635 640 Gly Asn Phe Lys Gln
Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655 Glu Gly Phe
Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670 Tyr
Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680
685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met
690 695 700 Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Arg Phe Asn His
Ile Ser705 710 715 720 Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala
Asp Ala Ile Lys Ala 725 730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr
Ser Gly Ser Asp Lys Glu Asn 740 745 750 Ile Lys Ser Gln Val Glu Asn
Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765 Ser Glu Ala Met Asn
Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780 Thr Tyr Leu
Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn785 790 795 800
Lys Phe Asp Leu Lys Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805
810 815 His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys
Val 820 825 830 Asn Glu Ser Phe Glu Asn Thr Ile Pro Phe Asn Ile Phe
Ser Tyr Thr 835 840 845 Asn Asn Ser Leu Leu Lys Asp Met Ile Asn Glu
Tyr Phe Asn Ser Ile 850 855 860 Asn Asp Ser Lys Ile Leu Ser Leu Gln
Asn Lys Lys Asn Thr Leu Met865 870 875 880 Asp Thr Ser Gly Tyr Asn
Ala Glu Val Arg Val Glu Gly Asn Val Gln 885 890 895 Leu Asn Pro Ile
Phe Pro Phe Asp Phe Lys Leu Gly Ser Ser Gly Asp 900 905 910 Asp Arg
Gly Lys Val Ile Val Thr Gln Asn Glu Asn Ile Val Tyr Asn 915 920 925
Ala Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile Arg Ile Asn Lys 930
935 940 Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp Ser Val Lys
Asn945 950 955 960 Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe
Leu Val Phe Thr 965 970 975 Leu Lys Gln Asn Glu Asn Ser Glu Gln Asp
Ile Asn Phe Ser Tyr Asp 980 985 990 Ile Ser Lys Asn Ala Ala Gly Tyr
Asn Lys Trp Phe Phe Val Thr Ile 995 1000 1005 Thr Thr Asn Met Met
Gly Asn Met Met Ile Tyr Ile Asn Gly Lys Leu 1010 1015 1020 Ile Asp
Thr Ile Lys Val Lys Glu Leu Thr Gly Ile Asn Phe Ser Lys1025 1030
1035 1040Thr Ile Thr Phe Gln Met Asn Lys Ile Pro Asn Thr Gly Leu
Ile Thr 1045 1050 1055 Ser Asp Ser Asp Asn Ile Asn Met Trp Ile Arg
Asp Phe Tyr Ile Phe 1060 1065 1070 Ala Lys Glu Leu Asp Asp Lys Asp
Ile Asn Ile Leu Phe Asn Ser Leu 1075 1080 1085 Gln Tyr Thr Asn Val
Val Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr 1090 1095 1100 Asp Lys
Glu Tyr Tyr Met Ile Asn Val Asn Tyr Met Asn Arg Tyr Met1105 1110
1115 1120Ser Lys Lys Gly Asn Gly Ile Val Phe Asn Thr Arg Lys Asn
Asn Asn 1125 1130 1135 Asp Phe Asn Glu Gly Tyr Lys Ile Ile Ile Lys
Arg Ile Arg Gly Asn 1140 1145 1150 Thr Asn Asp Thr Arg Val Arg Gly
Glu Asn Val Leu Tyr Phe Asn Thr 1155 1160 1165 Thr Ile Asp Asn Lys
Gln Tyr Ser Leu Gly Met Tyr Lys Pro Ser Arg 1170 1175 1180 Asn Leu
Gly Thr Asp Leu Val Pro Leu Gly Ala Leu Asp Gln Pro Met1185 1190
1195 1200Asp Glu Ile Arg Lys Tyr Gly Ser Phe Ile Ile Gln Pro Cys
Asn Thr 1205 1210 1215 Phe Asp Tyr Tyr Ala Ser Gln Leu Phe Leu Ser
Ser Asn Ala Thr Thr 1220 1225 1230 Asn Arg Leu Gly Ile Leu Ser Ile
Gly Ser Tyr Ser Phe Lys Leu Gly 1235 1240 1245 Asp Asp Tyr Trp Phe
Asn His Glu Tyr Leu Ile Pro Val Ile Lys Ile 1250 1255 1260 Glu His
Tyr Ala Ser Leu Leu Glu Ser Thr Ser Thr His Trp Val Phe1265 1270
1275 1280Val Pro Ala Ser Glu 1285151252PRTClostridium botulinum E1
15Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg1
5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys
Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg
Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr
Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr
Leu Gln Ser Asp Glu Glu Lys65 70 75 80 Asp Arg Phe Leu Lys Ile Val
Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn Leu Ser Gly Gly
Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly
Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125 Ala
Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135
140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu
Thr145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met
Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe
Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Asn Asp Asn Ser Met Asn
Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu Met His Glu Leu
Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys Gly Ile Thr
Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225 230 235 240 Ile
Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250
255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr
260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu
Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys
Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser
Gly Ile Tyr Ser Val Asn305 310 315 320 Ile Asn Lys Phe Asn Asp Ile
Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu Ala Thr
Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr
Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 Tyr
Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375
380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr
Pro Ile Thr385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg
Phe Cys Lys Asn Ile Val 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser
Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu Phe Phe Val Ala
Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn Thr Pro Lys
Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu Asn
Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala465 470 475
480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala
485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu
Gln His 500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp
Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr
Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr
Thr Phe Phe Ser Ser Glu Phe Ile545 550 555 560 Asn Asn Val Asn Lys
Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 Gln Gln Val
Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 Val
Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600
605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala
610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro
Glu Leu625 630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys
Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys
Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Lys
Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp Met Thr Lys Ile
Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 Tyr Gln Ala
Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu705 710 715 720
Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725
730 735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val
Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser
Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile
Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu
Leu Asn Tyr Ile Ile Gln His785 790 795 800 Gly Ser Ile Leu Gly Glu
Ser Gln Gln Glu Leu Asn Ser Met Val Thr 805 810 815 Asp Thr Leu Asn
Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 Asp Lys
Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850
855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr
Lys865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn
Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile
Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe Ser Ile Ser Phe Trp
Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys Ile Val Asn Val Asn
Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 Asp Asn Asn Ser
Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile945 950 955 960 Trp
Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970
975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe
980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr
Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn
Leu Gly Asn Ile His 1010 1015 1020 Val Ser Asp Asn Ile Leu Phe Lys
Ile Val Asn Cys Ser Tyr Thr Arg1025 1030 1035 1040Tyr Ile Gly Ile
Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr
Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu 1060
1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr
Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp
Arg Arg Lys Asp Ser 1090 1095 1100 Thr Leu Ser Ile Asn Asn Ile Arg
Ser Thr Ile Leu Leu Ala Asn Arg1105 1110 1115 1120Leu Tyr Ser Gly
Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 Thr
Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140
1145 1150 Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr
Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser
Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln Val Val Val Met Asn Ser
Val Gly Asn Asn Cys Thr Met Asn1185 1190 1195 1200Phe Lys Asn Asn
Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205 1210 1215 Asp
Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220
1225 1230 Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu
His Gly 1235 1240 1245 Trp Gln Glu Lys 1250 161252PRTClostridium
botulinum E2 16Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val
Asn Asp Arg1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln
Glu Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile
Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln Asp Phe
His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr
Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys65 70 75 80 Asp Arg Phe
Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn
Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105
110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp
115 120 125 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ile Gln Asp
Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp
Leu Phe Glu Thr145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn
Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala Ile
Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Asn Asp Asn
Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu Met
His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys
Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225 230
235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe
Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn
Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala
Ser Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn
Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys
Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310 315 320 Ile Asn Lys Phe
Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp
Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350
Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355
360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn
Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr
Pro Ile Thr385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg
Phe Cys Lys Asn Ile Val 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser
Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu Phe Phe Val Ala
Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn Thr Pro Lys
Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu Asn
Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala465 470 475
480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala
485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu
Gln His 500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp
Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr
Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr
Thr Phe Phe Ser Ser Glu Phe Ile545 550 555 560 Asn Asn Val Asn Lys
Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 Gln Gln Val
Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 Val
Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600
605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala
610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro
Glu Leu625 630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys
Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys
Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Lys
Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp Met Thr Lys Ile
Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 Tyr Gln Ala
Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu705 710 715 720
Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725
730 735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val
Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser
Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile
Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu
Leu Asn Tyr Ile Ile Gln His785 790 795 800 Gly Ser Ile Leu Gly Glu
Ser Gln Gln Glu Leu Asn Ser Met Val Thr 805 810 815 Asp Thr Leu Asn
Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 Asp Lys
Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850
855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr
Lys865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn
Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile
Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe Ser Ile Ser Phe Trp
Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys Ile Val Asn Val Asn
Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 Asp Asn Asn Ser
Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile945 950 955 960 Trp
Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970
975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe
980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr
Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn
Leu Gly Asn Ile His 1010 1015 1020 Val Ser Asp Asn Ile Leu Phe Lys
Ile Val Asn Cys Ser Tyr Thr Arg1025 1030 1035 1040Tyr Ile Gly Ile
Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr
Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu 1060
1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr
Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp
Arg Arg Thr Asp Ser 1090 1095 1100 Thr Leu Ser Ile Asn Asn Ile Arg
Ser Thr Ile Leu Leu Ala Asn Arg1105 1110 1115 1120Leu Tyr Ser Gly
Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 Thr
Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140
1145 1150 Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr
Asn Thr 1155 1160 1165 Thr Asn Lys Glu Lys Thr Ile Lys Ser Ser Ser
Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln Val Val Val Met Asn Ser
Val Gly Asn Asn Cys Thr Met Asn1185 1190 1195 1200Phe Lys Asn Asn
Asn Gly Asn Asn Ile Gly Met Leu Gly Phe Lys Asp 1205 1210 1215 Asn
Thr Leu Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn 1220
1225 1230 Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu
His Gly 1235 1240 1245 Trp Gln Glu Lys 1250 171252PRTClostridium
botulinum E3 17Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val
Asn Asp Arg1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln
Glu Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile
Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln Asp Phe
His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr
Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys65 70 75 80 Asp Arg Phe
Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn
Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105
110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp
115 120 125 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln His
Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp
Leu Phe Glu Thr145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn
Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala Ile
Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185
190 Arg Phe Asn Asp Asn Ser Ile Asn Glu Phe Ile Gln Asp Pro Ala Leu
195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr
Gly Ala 210 215 220 Lys Gly Ile Thr Thr Thr Cys Ile Ile Thr Gln Gln
Gln Asn Pro Leu225 230 235 240 Ile Thr Asn Arg Lys Gly Ile Asn Ile
Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Asn Asp Leu Asn Ile Ile
Thr Val Ala Gln Tyr Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Asn
Asp Tyr Arg Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val
Ser Asn Pro Gln Leu Asn Pro Tyr Lys Asp Ile Phe Gln 290 295 300 Glu
Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310
315 320 Ile Asn Lys Phe Asp Asp Ile Leu Lys Lys Leu Tyr Ser Phe Thr
Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Glu
Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu
Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile
Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu
Asn Pro Arg Ile Ile Lys Pro Ile Thr385 390 395 400 Gly Arg Gly Leu
Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val
Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435
440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn
Tyr 450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser
Glu Ser Ala465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu
Thr Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn
Gly Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn
Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu
Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu
Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550 555
560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile
565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys
Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro
Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys
Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile
Leu Leu Glu Phe Glu Pro Glu Leu625 630 635 640 Leu Ile Pro Thr Ile
Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn
Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu
Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680
685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met
690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile
Ile Glu705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys
Asn Glu Leu Thr Asn 725 730 735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn
Glu Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp
Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu
Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu
Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His785 790 795 800
Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr 805
810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr
Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys
Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn
Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn
Ile Asn Gly Asp Val Tyr Lys865 870 875 880 Tyr Pro Thr Asn Lys Asn
Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile
Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn
Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925
Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930
935 940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile
Ile945 950 955 960 Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys
Leu Ala Phe Asn 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr
Ile Asn Lys Trp Ile Phe 980 985 990 Val Thr Ile Thr Asn Asp Arg Leu
Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp
Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 Val Ser
Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg1025 1030
1035 1040Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu
Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro
Asn Thr Asn Ile Leu 1060 1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu
Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys
Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser 1090 1095 1100 Thr Leu
Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg1105 1110
1115 1120Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn
Ser Ser 1125 1130 1135 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln
Val Tyr Ile Asn Phe 1140 1145 1150 Val Ala Ser Lys Thr His Leu Phe
Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys
Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln
Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn1185 1190
1195 1200Phe Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe
Lys Ala 1205 1210 1215 Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr
His Met Arg Asp His 1220 1225 1230 Thr Asn Ser Asn Gly Cys Phe Trp
Asn Phe Ile Ser Glu Glu His Gly 1235 1240 1245 Trp Gln Glu Lys 1250
181274PRTClostridium botulinum F1 18Met Pro Val Ala Ile Asn Ser Phe
Asn Tyr Asn Asp Pro Val Asn Asp1 5 10 15 Asp Thr Ile Leu Tyr Met
Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr Tyr Lys Ala
Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg Asn
Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60
Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr65
70 75 80 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu
Phe Lys 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu
Gln Glu Ile Ser 100 105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His
Thr Pro Ile Asp Glu Phe 115 120 125 Ser Pro Val Thr Arg Thr Thr Ser
Val Asn Ile Lys Leu Ser Thr Asn 130 135 140 Val Glu Ser Ser Met Leu
Leu Asn Leu Leu Val Leu Gly Ala Gly Pro145 150 155 160 Asp Ile Phe
Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170 175 Asp
Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile 180 185
190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly
195 200 205 Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala
Ile Ser 210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu
Tyr Gly Ala Arg225 230 235 240 Gly Val Thr Tyr Glu Glu Thr Ile Glu
Val Lys Gln Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg
Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu Asn Ile
Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 Asn Leu Leu
Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295 300 Asn
Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe305 310
315 320 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr
Val 325 330 335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr
Ser Phe Thr 340 345 350 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys
Cys Arg Asn Thr Tyr 355 360 365 Phe Ile Lys Tyr Glu Phe Leu Lys Val
Pro Asn Leu Leu Asp Asp Asp 370 375 380 Ile Tyr Thr Val Ser Glu Gly
Phe Asn Ile Gly Asn Leu Ala Val Asn385 390 395 400 Asn Arg Gly Gln
Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415 Pro Asp
Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430
Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435
440 445 Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn
Glu 450 455 460 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr
Asn Leu Asn465 470 475 480 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val
Ile Leu Asp Tyr Asn Ser 485 490 495 Gln Thr Ile Pro Gln Ile Ser Asn
Arg Thr Leu Asn Thr Leu Val Gln 500 505 510 Asp Asn Ser Tyr Val Pro
Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 Glu Glu Tyr Asp
Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540 Gln Lys
Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile545 550 555
560 Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu
565 570 575 Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe
Ile Asp 580 585 590 Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu
Ala Thr Gln Lys 595 600 605 Ser Thr Val Asp Lys Ile Ala Asp Ile Ser
Leu Ile Val Pro Tyr Val 610 615 620 Gly Leu Ala Leu Asn Ile Ile Ile
Glu Ala Glu Lys Gly Asn Phe Glu625 630 635 640 Glu Ala Phe Glu Leu
Leu Gly Val Gly Ile Leu Leu Glu Phe Val Pro 645 650 655 Glu Leu Thr
Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile 660 665 670 Asp
Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser 675 680
685 Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val
690 695 700 Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg
Lys Glu705 710 715 720 Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp
Ala Ile Lys Thr Ala 725 730 735 Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr
Ser Asp Glu Lys Asn Arg Leu 740 745 750 Glu Ser Glu Tyr Asn Ile Asn
Asn Ile Glu Glu Glu Leu Asn Lys Lys 755 760 765 Val Ser Leu Ala Met
Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 780 Ile Ser Tyr
Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu785 790 795 800
Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805
810 815 Asp His Arg Ser Ile Leu Gly Glu Gln Thr Asn Glu Leu Ser Asp
Leu 820 825 830 Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu
Ser Ser Tyr 835 840 845 Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn
Arg Leu Tyr Lys Lys 850 855 860 Ile Lys Asp Ser Ser Ile Leu Asp Met
Arg Tyr Glu Asn Asn Lys Phe865 870 875 880 Ile Asp Ile Ser Gly Tyr
Gly Ser Asn Ile Ser Ile Asn Gly Asn Val 885 890 895 Tyr Ile Tyr Ser
Thr Asn Arg Asn Gln Phe Gly Ile Tyr Asn Ser Arg 900 905 910 Leu Ser
Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Ser 915 920 925
Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys His 930
935 940 Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile Asn Cys
Met945 950 955 960 Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg
Thr Val Arg Asp 965 970 975 Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr
Ser Gly Asn Lys Glu Asn 980 985 990 Leu Ile Phe Arg Tyr Glu Glu Leu
Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005 Lys Trp Ile Phe Val
Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 1010 1015 1020 Ile Tyr
Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn Leu1025 1030
1035 1040Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val
Gly Cys 1045 1050 1055 Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe
Lys Val Phe Asn Thr 1060 1065 1070 Glu Leu Asp Lys Thr Glu Ile Glu
Thr Leu Tyr Ser Asn Glu Pro Asp 1075 1080 1085 Pro Ser Ile Leu Lys
Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 1090 1095 1100 Lys Tyr
Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr Ile Thr Leu1105 1110
1115 1120Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg Gly Val Thr
Glu Gly 1125 1130 1135 Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly
Val Glu Val Ile Ile 1140 1145 1150 Arg Lys Asn Gly Pro Ile Asp Ile
Ser Asn Thr Asp Asn Phe Val Arg 1155 1160 1165 Lys Asn Asp Leu Ala
Tyr Ile Asn Val Val Asp Arg Gly Val Glu Tyr 1170 1175 1180 Arg Leu
Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys Ile Ile Arg Thr1185 1190
1195 1200Ser Asn Leu Asn Asp Ser Leu Gly Gln Ile Ile Val Met Asp
Ser Ile 1205 1210 1215 Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn
Asn Gly Ser Asn Ile 1220 1225 1230 Gly Leu Leu Gly Phe His Ser Asn
Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245 Tyr
Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250
1255 1260 Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu1265 1270
191280PRTClostridium botulinum F2 19Met Pro Val Val Ile Asn Ser Phe
Asn Tyr Asn Asp Pro Val Asn Asp1 5 10 15 Glu Thr Ile Leu Tyr Met
Gln Lys Pro Tyr Glu Glu Arg Ser Arg Lys 20 25 30 Tyr Tyr Lys Ala
Phe Glu Ile Met Pro Asn Val Trp Ile Met Pro Glu 35 40 45 Arg Asp
Thr Ile Gly Thr Lys Pro Asp Glu Phe Gln Val Pro Asp Ser 50 55 60
Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr65
70 75 80 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Met Ile Lys Leu
Phe Asn 85 90 95 Arg Ile Asn Ser Asn Pro Thr Gly Lys Val Leu Leu
Glu Glu Val Ser 100 105 110 Asn Ala Arg Pro Tyr Leu Gly Asp Asp Asp
Thr Leu Ile Asn Glu Phe 115 120 125 Leu Pro Val Asn Val Thr Thr Ser
Val Asn Ile Lys Phe Ser Thr Asp 130 135 140 Val Glu Ser Ser Ile Ile
Ser Asn Leu Leu Val Leu Gly Ala Gly Pro145 150 155 160 Asp Ile Phe
Lys Ala Tyr Cys Thr Pro Leu Val Arg Phe Asn Lys Ser 165 170 175 Asp
Lys Leu Ile Glu Pro Ser Asn His Gly Phe Gly Ser Ile Asn Ile 180 185
190 Leu Thr Phe Ser Pro Glu Tyr Glu His Ile Phe Asn Asp Ile Ser Gly
195 200 205 Gly Asn His Asn Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala
Ile Ser 210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu
Tyr Gly Ala Lys225 230 235 240 Ala Val Thr His Lys Glu Ser Leu Val
Ala Glu Arg Gly Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg
Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 Glu Asp Leu Asn Ile
Ile Pro Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 Asp Leu Leu
Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Arg Glu Val 290 295 300 Asn
Thr Ala Pro Pro Gly Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe305 310
315 320 Gln Trp Lys Tyr Gly Leu Asp Arg Asn Ala Asp Gly Ser Tyr Thr
Val 325 330 335 Asn Arg Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr
Ser Phe Thr 340 345 350 Glu Ile Asp Leu Ala Asn Lys Phe Lys Val Lys
Cys Arg Asn Thr Tyr 355 360 365 Phe Ile Lys Tyr Gly Phe Val Lys Val
Pro Asn Leu Leu Asp Asp Asp 370 375 380 Ile Tyr Thr Val Ser Glu Gly
Phe Asn Ile Gly Asn Leu Ala Val Asn385 390 395 400 Asn Arg Gly Gln
Asn Ile Asn Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415 Pro Asp
Lys Gly Leu Val Glu Lys Ile Ile Lys Phe Cys Lys Ser Ile 420 425 430
Ile Pro Arg Lys Gly Thr Lys Gln Ser Pro Ser Leu Cys Ile Arg Val 435
440 445 Asn Asn Arg Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn
Glu 450 455 460 Ser Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr
Asn Leu Asn465 470 475 480 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val
Ile Leu Asp Tyr Asn Ser 485 490 495 Glu Thr Ile Pro Gln Ile Ser Asn
Arg Thr Leu Asn Thr Leu Val Gln 500 505 510 Asp Asn Ser Tyr Val Pro
Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 Glu Glu Tyr Asp
Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540 Gln Lys
Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile545 550 555
560 Asp Thr Ala Leu Leu Glu Glu Ser Lys Val Tyr Thr Phe Phe Ser Ser
565 570 575 Glu Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu
Phe Ile 580 585 590 Asp Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr
Glu Ala Thr Gln 595 600 605 Lys Ser Thr Val Asp Lys Ile Ala Asp Ile
Ser Leu Ile Val Pro Tyr 610 615 620 Val Gly Leu Ala Leu Asn Ile Val
Ile Glu Ala Glu Lys Gly Asn Phe625 630 635 640 Glu Glu Ala Phe Glu
Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Val 645 650 655 Pro Glu Leu
Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr 660 665 670 Ile
Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn 675 680
685 Ser Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile
690 695 700 Val Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys
Arg Lys705 710 715 720 Glu Gln Met Tyr Gln Ala Leu Gln Asn Gln Val
Asp Ala Ile Lys Thr 725 730 735 Ala Ile Glu Tyr Lys Tyr Asn Asn Tyr
Thr Ser Asp Glu Lys Asn Arg 740 745 750 Leu Glu Ser Lys Tyr Asn Ile
Asn Asn Ile Glu Glu Glu Leu Asn Lys 755 760 765 Lys Val Ser Leu Ala
Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser 770 775 780 Ser Ile Ser
Tyr Leu Met Lys Leu Ile Asn Glu Ala Glu Val Gly Lys785 790 795 800
Leu Lys Glu Tyr Asp Lys His Val Lys Ser Asp Leu Leu Asp Tyr Ile 805
810 815 Leu Tyr His Lys Leu Ile Leu Gly Glu Gln Thr Lys Glu Leu Ile
Asp 820 825 830 Leu Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu
Leu Ser Ser 835 840 845 Tyr Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe
Asn Arg Leu Tyr Lys 850 855 860 Lys Ile Lys Asp Ser Ser Ile Leu Asp
Met Arg Tyr Glu Asn Asn Lys865 870 875 880 Phe Ile Asp Ile Ser Gly
Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn 885 890 895 Val Tyr Ile Tyr
Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Ser Gly 900 905 910 Arg Leu
Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn 915 920 925
Ser Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Thr Ile Pro Lys 930
935 940 His Tyr Arg Pro Met Asn Arg Asn Arg Glu Tyr Thr Ile Ile Asn
Cys945 950 955 960 Met Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu
Arg Thr Ile Arg 965 970 975 Asp Cys Glu Ile Ile Trp Thr Leu Gln Asp
Thr Ser Gly Asn Lys Glu 980 985 990 Lys Leu Ile Phe Arg Tyr Glu Glu
Leu Ala Ser Ile Ser Asp Tyr Ile 995 1000 1005 Asn Lys Trp Ile Phe
Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser 1010 1015 1020 Arg Ile
Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn1025 1030
1035 1040Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile
Val Gly 1045 1050 1055 Cys Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr
Phe Lys Val Phe Asn 1060 1065 1070 Thr Glu Leu Asp Lys Thr Glu Ile
Glu Thr Leu Tyr Ser Asn Glu Pro 1075 1080 1085 Asp Pro Ser Ile Leu
Lys Asp Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn 1090 1095 1100 Lys Lys
Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr Ile Thr1105 1110
1115 1120Arg Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg Gly Val
Thr Gly 1125 1130 1135 Gly Ile Ser Val Phe Leu Asn Tyr Lys Leu Tyr
Glu Gly Val Glu Val 1140 1145 1150 Ile Ile Arg Lys Asn Ala Pro Ile
Asp Ile Ser Asn Thr Asp Asn Phe 1155 1160 1165 Val Arg Lys Asn Asp
Leu Ala Tyr Ile Asn Val Val Asp His Gly Val 1170 1175 1180 Glu Tyr
Arg Leu Tyr Ala Asp Ile Ser Ile Thr Lys Ser Glu Lys Ile1185 1190
1195 1200Ile Lys Leu Ile Arg Thr Ser Asn Pro Asn Asp Ser Leu Gly
Gln Ile 1205 1210 1215 Ile Val Met Asp Ser Ile Gly Asn Asn Cys Thr
Met Asn Phe Gln Asn 1220 1225 1230 Asn Asp Gly Ser Asn Ile Gly Leu
Leu Gly Phe His Ser Asp Asp Leu 1235 1240 1245 Val Ala Ser Ser Trp
Tyr Tyr Asn His Ile Arg Arg Asn Thr Ser Ser 1250 1255 1260 Asn Gly
Cys Phe Trp Ser Phe Ile Ser Lys Glu His Gly Trp Lys Glu1265 1270
1275 1280201278PRTClostridium botulinum F3 20Met Pro Val Val Ile
Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp1 5 10 15 Asp Thr Ile
Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr
Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40
45 Arg Asn Thr Ile Gly Thr Asp Pro Ser Asp Phe Asp Pro Pro Ala Ser
50 55 60 Leu Glu Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu
Thr Thr65 70 75 80 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile
Lys Leu Phe Lys 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Glu Val
Leu Leu Gln Glu Ile Ser 100 105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn
Glu His Thr Pro Ile Asn Glu Phe 115 120 125 His Pro Val Thr Arg Thr
Thr Ser Val Asn Ile Lys Ser Ser Thr Asn 130 135 140 Val Lys Ser Ser
Ile Ile Leu Asn Leu Leu Val Leu Gly Ala Gly Pro145 150 155 160 Asp
Ile Phe Glu Asn Ser Ser Tyr Pro Val Arg Lys Leu Met Asp Ser 165 170
175 Gly Gly Val Tyr Asp Pro Ser Asn Asp Gly Phe Gly Ser Ile Asn Ile
180 185 190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile
Ser Gly 195 200 205 Gly Tyr Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp
Pro Ala Ile Ser 210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His
Gly Leu Tyr Gly Ala Arg225 230 235 240 Gly Val Thr Tyr Lys Glu Thr
Ile Lys Val Lys Gln Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro
Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu
Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 Asn
Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Arg Val 290 295
300 Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr
Phe305 310 315 320 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly
Ser Tyr Thr Val 325 330 335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys
Lys Leu Tyr Ser Phe Thr 340 345 350 Glu Ile Asp Leu Ala Asn Lys Phe
Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365 Phe Ile Lys Tyr Gly Phe
Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380 Ile Tyr Thr Val
Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn385 390 395 400 Asn
Arg Gly Gln Asn Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410
415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val
420 425 430 Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile
Arg Val 435 440 445 Asn Asn Arg Glu Leu Phe Phe Val Ala Ser Glu Ser
Ser Tyr Asn Glu 450 455 460 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp
Asp Thr Thr Asn Leu Asn465 470 475 480 Asn Asn Tyr Arg Asn Asn Leu
Asp Glu Val Ile Leu Asp Tyr Asn Ser 485 490 495 Glu Thr Ile Pro Gln
Ile Ser Asn Gln Thr Leu Asn Thr Leu Val Gln 500 505 510 Asp Asp Ser
Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 Glu
Glu His Asn Val Val Asp Leu Asn Val Phe Phe Tyr Leu His Ala 530 535
540 Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser
Ile545 550 555 560 Asp Thr Ala Leu Ser Glu Glu Ser Gln Val Tyr Thr
Phe Phe Ser Ser 565 570 575 Glu Phe Ile Asn Thr Ile Asn Lys Pro Val
His Ala Ala Leu Phe Ile 580 585 590 Ser Trp Ile Asn Gln Val Ile Arg
Asp Phe Thr Thr Glu Ala Thr Gln 595 600 605 Lys Ser Thr Phe Asp Lys
Ile Ala Asp Ile Ser Leu Val Val Pro Tyr 610 615 620 Val Gly Leu Ala
Leu Asn Ile Gly Asn Glu Val Gln Lys Glu Asn Phe625 630 635 640 Lys
Glu Ala Phe Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Val 645 650
655 Pro Glu Leu Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe
660 665 670 Ile Gly Ser Ser Glu Asn Lys Asn Lys Ile Ile Lys Ala Ile
Asn Asn 675 680 685 Ser Leu Met Glu Arg Glu Thr Lys Trp Lys Glu Ile
Tyr Ser Trp Ile 690 695 700 Val Ser Asn Trp Leu Thr Arg Ile Asn Thr
Gln Phe Asn Lys Arg Lys705 710 715 720 Glu Gln Met Tyr Gln Ala Leu
Gln Asn Gln Val Asp Ala Ile Lys Thr 725 730 735 Val Ile Glu Tyr Lys
Tyr Asn Asn Tyr Thr Ser Asp Glu Arg Asn Arg 740 745 750 Leu Glu Ser
Glu Tyr Asn Ile Asn Asn Ile Arg Glu Glu Leu Asn Lys 755 760 765 Lys
Val Ser Leu Ala Met Glu Asn Ile Glu Arg Phe Ile Thr Glu Ser 770 775
780 Ser Ile Phe Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Ser
Lys785 790 795 800 Leu Arg Glu Tyr Asp Glu Gly Val Lys Glu Tyr Leu
Leu Asp Tyr Ile 805 810 815 Ser Glu His Arg Ser Ile Leu Gly Asn Ser
Val Gln Glu Leu Asn Asp 820 825 830 Leu Val Thr Ser Thr Leu Asn Asn
Ser Ile Pro Phe Glu Leu Ser Ser 835 840 845 Tyr Thr Asn Asp Lys Ile
Leu Ile Leu Tyr Phe Asn Lys Leu Tyr Lys 850 855 860 Lys Ile Lys Asp
Asn Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys865 870 875 880 Phe
Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asp 885 890
895 Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Ser Ser
900 905 910 Lys Pro Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile
Tyr Asn 915 920 925 Gly Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val
Arg Ile Pro Lys 930 935 940 Tyr Phe Asn Lys Val Asn Leu Asn Asn Glu
Tyr Thr Ile Ile Asp Cys945 950 955 960 Ile Arg Asn Asn Asn Ser Gly
Trp Lys Ile Ser Leu Asn Tyr Asn Lys 965 970 975 Ile Ile Trp Thr Leu
Gln Asp Thr Ala Gly Asn Asn Gln Lys Leu Val 980 985 990 Phe Asn Tyr
Thr Gln Met Ile Ser Ile
Ser Asp Tyr Ile Asn Lys Trp 995 1000 1005 Ile Phe Val Thr Ile Thr
Asn Asn Arg Leu Gly Asn Ser Arg Ile Tyr 1010 1015 1020 Ile Asn Gly
Asn Leu Ile Asp Glu Lys Ser Ile Ser Asn Leu Gly Asp1025 1030 1035
1040Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys Asn Asp
1045 1050 1055 Thr Arg Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asp
Thr Glu Leu 1060 1065 1070 Gly Lys Thr Glu Ile Glu Thr Leu Tyr Ser
Asp Glu Pro Asp Pro Ser 1075 1080 1085 Ile Leu Lys Asp Phe Trp Gly
Asn Tyr Leu Leu Tyr Asn Lys Arg Tyr 1090 1095 1100 Tyr Leu Leu Asn
Leu Leu Arg Thr Asp Lys Ser Ile Thr Gln Asn Ser1105 1110 1115
1120Asn Phe Leu Asn Ile Asn Gln Gln Arg Gly Val Tyr Gln Lys Pro Asn
1125 1130 1135 Ile Phe Ser Asn Thr Arg Leu Tyr Thr Gly Val Glu Val
Ile Ile Arg 1140 1145 1150 Lys Asn Gly Ser Thr Asp Ile Ser Asn Thr
Asp Asn Phe Val Arg Lys 1155 1160 1165 Asn Asp Leu Ala Tyr Ile Asn
Val Val Asp Arg Asp Val Glu Tyr Arg 1170 1175 1180 Leu Tyr Ala Asp
Ile Ser Ile Ala Lys Pro Glu Lys Ile Ile Lys Leu1185 1190 1195
1200Ile Arg Thr Ser Asn Ser Asn Asn Ser Leu Gly Gln Ile Ile Val Met
1205 1210 1215 Asp Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn
Asn Asn Gly 1220 1225 1230 Gly Asn Ile Gly Leu Leu Gly Phe His Ser
Asn Asn Leu Val Ala Ser 1235 1240 1245 Ser Trp Tyr Tyr Asn Asn Ile
Arg Lys Asn Thr Ser Ser Asn Gly Cys 1250 1255 1260 Phe Trp Ser Phe
Ile Ser Lys Glu His Gly Trp Gln Glu Asn1265 1270 1275
211297PRTClostridium botulinum GVARIANT7Identity of Xaa at position
7 is unknown 21Met Pro Val Asn Ile Lys Xaa Phe Asn Tyr Asn Asp Pro
Ile Asn Asn1 5 10 15 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp
Pro Gly Pro Gly Thr 20 25 30 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp
Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Phe Thr Tyr Gly Phe Gln
Pro Asp Gln Phe Asn Ala Ser Thr Gly 50 55 60 Val Phe Ser Lys Asp
Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys65 70 75 80 Thr Asp Ala
Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 90 95 Asn
Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile 100 105
110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys
115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile
Ile Gln 130 135 140 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr
Asn Leu Ile Ile145 150 155 160 Phe Gly Pro Gly Pro Val Leu Ser Asp
Asn Phe Thr Asp Ser Met Ile 165 170 175 Met Asn Gly His Ser Pro Ile
Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190 Ile Arg Phe Cys Pro
Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Asp
Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220 Ala
Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230
235 240 Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu
Phe 245 250 255 Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu
Tyr Thr Phe 260 265 270 Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser
Thr Asp Met Asn Ile 275 280 285 Tyr Asn Lys Ala Leu Gln Asn Phe Gln
Asp Ile Ala Asn Arg Leu Asn 290 295 300 Ile Val Ser Ser Ala Gln Gly
Ser Gly Ile Asp Ile Ser Leu Tyr Lys305 310 315 320 Gln Ile Tyr Lys
Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330 335 Tyr Ser
Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 345 350
Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr 355
360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu
Lys 370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe
Asn Ile Ala385 390 395 400 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly
Gln Asn Lys Ala Val Asn 405 410 415 Lys Glu Ala Tyr Glu Glu Ile Ser
Leu Glu His Leu Val Ile Tyr Arg 420 425 430 Ile Ala Met Cys Lys Pro
Val Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445 Gln Cys Ile Ile
Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys 450 455 460 Asp Ser
Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn465 470 475
480 Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile
485 490 495 Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu
Asn Thr 500 505 510 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro
Val Tyr Ile Lys 515 520 525 Gln Ser Ala Leu Lys Lys Ile Phe Val Asp
Gly Asp Ser Leu Phe Glu 530 535 540 Tyr Leu His Ala Gln Thr Phe Pro
Ser Asn Ile Glu Asn Leu Gln Leu545 550 555 560 Thr Asn Ser Leu Asn
Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr 565 570 575 Phe Phe Ser
Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590 Ser
Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser 595 600
605 Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Val Ser Ile
610 615 620 Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu
Thr Ala625 630 635 640 Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly
Gly Ala Ala Ile Leu 645 650 655 Met Glu Phe Ile Pro Glu Leu Ile Val
Pro Ile Val Gly Phe Phe Thr 660 665 670 Leu Glu Ser Tyr Val Gly Asn
Lys Gly His Ile Ile Met Thr Ile Ser 675 680 685 Asn Ala Leu Lys Lys
Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700 Ile Val Ser
Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile705 710 715 720
Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu 725
730 735 Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys
Met 740 745 750 Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu
Asn Gln Ser 755 760 765 Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe
Ile Asn Gln Cys Ser 770 775 780 Ile Ser Tyr Leu Met Asn Arg Met Ile
Pro Leu Ala Val Lys Lys Leu785 790 795 800 Lys Asp Phe Asp Asp Asn
Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp 805 810 815 Thr Asn Glu Leu
Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys 820 825 830 Val Asn
Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr 835 840 845
Thr Lys Asp Thr Ile Leu Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn 850
855 860 Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg
Leu865 870 875 880 Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val
Gly Ser Asp Val 885 890 895 Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe
Lys Leu Asn Asn Ser Glu 900 905 910 Asn Ser Asn Ile Thr Ala His Gln
Ser Lys Phe Val Val Tyr Asp Ser 915 920 925 Met Phe Asp Asn Phe Ser
Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940 Asn Asn Asn Asp
Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile945 950 955 960 Ser
Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970
975 Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile
980 985 990 Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile
Asn Lys 995 1000 1005 Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu
Gly Asn Ala Asn Ile 1010 1015 1020 Tyr Ile Asn Gly Ser Leu Lys Lys
Ser Glu Lys Ile Leu Asn Leu Asp1025 1030 1035 1040Arg Ile Asn Ser
Ser Asn Asp Ile Asp Phe Lys Leu Ile Asn Cys Thr 1045 1050 1055 Asp
Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg 1060
1065 1070 Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gln
Ser Ser 1075 1080 1085 Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro
Leu Arg Tyr Asp Thr 1090 1095 1100 Gln Tyr Tyr Leu Phe Asn Gln Gly
Met Gln Asn Ile Tyr Ile Lys Tyr1105 1110 1115 1120Phe Ser Lys Ala
Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn 1125 1130 1135 Asn
Ala Ala Ile Asn Tyr Gln Asn Leu Tyr Leu Gly Leu Arg Phe Ile 1140
1145 1150 Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn Asn Asp Asn
Ile Val 1155 1160 1165 Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp
Asn Ile Ser Asp Glu 1170 1175 1180 Ser Tyr Arg Val Tyr Val Leu Val
Asn Ser Lys Glu Ile Gln Thr Gln1185 1190 1195 1200Leu Phe Leu Ala
Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215 Gln
Ile Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu 1220
1225 1230 Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly
Lys Phe 1235 1240 1245 Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr
Asp Asn Tyr Phe Cys 1250 1255 1260 Ile Ser Gln Trp Tyr Leu Arg Arg
Ile Ser Glu Asn Ile Asn Lys Leu1265 1270 1275 1280Arg Leu Gly Cys
Asn Trp Gln Phe Ile Pro Val Asp Glu Gly Trp Thr 1285 1290 1295
Glu221315PRTClostridium tetani 22Met Pro Ile Thr Ile Asn Asn Phe
Arg Tyr Ser Asp Pro Val Asn Asn1 5 10 15 Asp Thr Ile Ile Met Met
Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala
Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr
Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60
Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr65
70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu
Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu
Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr
Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser
Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys
Ser Ala Met Leu Thr Asn Leu Ile Ile Phe145 150 155 160 Gly Pro Gly
Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg
Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185
190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn
195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys
Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile
His Val Leu His225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser
His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His
Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly
Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu
Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys
Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp305 310
315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp
Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile
Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu
Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Arg Leu Ser Tyr Phe Ser
Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp
Thr Ile Tyr Asn Asp Thr Glu Gly Phe385 390 395 400 Asn Ile Glu Ser
Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val
Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430
Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435
440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly
Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe
Ile Ala Glu465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln
Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn
Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu
Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val
Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala
Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile545 550 555
560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile
565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr
Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn
Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp
Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr
Ile Asp Lys Ile Ser Asp Val Ser625 630 635 640 Thr Ile Val Pro Tyr
Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly
Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu
Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680
685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile
690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val
Tyr Lys705 710 715 720 Leu Val Lys Ala Lys
Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr
Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys
Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760
765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu
770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg
Glu Ser785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn
Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys
Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile
Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys
Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu
Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile865 870 875 880
Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885
890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp
Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu
Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met
Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser
Phe Trp Leu Arg Val Pro Lys945 950 955 960 Val Ser Ala Ser His Leu
Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met
Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser
Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000
1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe
Asn 1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile
Thr Asn Asp Arg1025 1030 1035 1040Leu Ser Ser Ala Asn Leu Tyr Ile
Asn Gly Val Leu Met Gly Ser Ala 1045 1050 1055 Glu Ile Thr Gly Leu
Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu 1060 1065 1070 Lys Leu
Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys 1075 1080
1085 Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys
Leu 1090 1095 1100 Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp
Phe Trp Gly Asn1105 1110 1115 1120Pro Leu Arg Tyr Asp Thr Glu Tyr
Tyr Leu Ile Pro Val Ala Ser Ser 1125 1130 1135 Ser Lys Asp Val Gln
Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr 1140 1145 1150 Asn Ala
Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg 1155 1160
1165 Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn
Asn 1170 1175 1180 Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile
Lys Leu Tyr Val1185 1190 1195 1200Ser Tyr Asn Asn Asn Glu His Ile
Val Gly Tyr Pro Lys Asp Gly Asn 1205 1210 1215 Ala Phe Asn Asn Leu
Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro 1220 1225 1230 Gly Ile
Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240
1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala
Ser 1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly
Asn Asp Pro Asn1265 1270 1275 1280Arg Asp Ile Leu Ile Ala Ser Asn
Trp Tyr Phe Asn His Leu Lys Asp 1285 1290 1295 Lys Ile Leu Gly Cys
Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp 1300 1305 1310 Thr Asn
Asp 1315231268PRTClostridium baratii 23Met Pro Val Asn Ile Asn Asn
Phe Asn Tyr Asn Asp Pro Ile Asn Asn1 5 10 15 Thr Thr Ile Leu Tyr
Met Lys Met Pro Tyr Tyr Glu Asp Ser Asn Lys 20 25 30 Tyr Tyr Lys
Ala Phe Glu Ile Met Asp Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg
Asn Ile Ile Gly Lys Lys Pro Ser Asp Phe Tyr Pro Pro Ile Ser 50 55
60 Leu Asp Ser Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr
Thr65 70 75 80 Asp Ala Glu Lys Asp Arg Phe Leu Lys Thr Val Ile Lys
Leu Phe Asn 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Gln Val Leu
Leu Glu Glu Ile Lys 100 105 110 Asn Gly Lys Pro Tyr Leu Gly Asn Asp
His Thr Ala Val Asn Glu Phe 115 120 125 Cys Ala Asn Asn Arg Ser Thr
Ser Val Glu Ile Lys Glu Ser Asn Gly 130 135 140 Thr Thr Asp Ser Met
Leu Leu Asn Leu Val Ile Leu Gly Pro Gly Pro145 150 155 160 Asn Ile
Leu Glu Cys Ser Thr Phe Pro Val Arg Ile Phe Pro Asn Asn 165 170 175
Ile Ala Tyr Asp Pro Ser Glu Lys Gly Phe Gly Ser Ile Gln Leu Met 180
185 190 Ser Phe Ser Thr Glu Tyr Glu Tyr Ala Phe Asn Asp Asn Thr Asp
Leu 195 200 205 Phe Ile Ala Asp Pro Ala Ile Ser Leu Ala His Glu Leu
Ile His Val 210 215 220 Leu His Gly Leu Tyr Gly Ala Lys Gly Val Thr
Asn Lys Lys Val Ile225 230 235 240 Glu Val Asp Gln Gly Ala Leu Met
Ala Ala Glu Lys Asp Ile Lys Ile 245 250 255 Glu Glu Phe Ile Thr Phe
Gly Gly Gln Asp Leu Asn Ile Ile Thr Asn 260 265 270 Ser Thr Asn Gln
Lys Ile Tyr Val Ile Leu Leu Ser Asn Tyr Thr Ala 275 280 285 Ile Ala
Ser Arg Leu Ser Gln Val Asn Arg Asn Asn Ser Ala Leu Asn 290 295 300
Thr Thr Tyr Tyr Lys Asn Phe Phe Gln Trp Lys Tyr Gly Leu Asp Gln305
310 315 320 Asp Ser Asn Gly Asn Tyr Thr Val Asn Ile Ser Lys Phe Asn
Ala Ile 325 330 335 Tyr Lys Lys Leu Phe Ser Phe Thr Glu Cys Asp Leu
Ala Gln Lys Phe 340 345 350 Gln Val Lys Asn Arg Ser Asn Tyr Leu Phe
His Phe Lys Pro Phe Arg 355 360 365 Leu Leu Asp Leu Leu Asp Asp Asn
Ile Tyr Ser Ile Ser Glu Gly Phe 370 375 380 Asn Ile Gly Ser Leu Arg
Val Asn Asn Asn Gly Gln Asn Ile Asn Leu385 390 395 400 Asn Ser Arg
Ile Val Gly Pro Ile Pro Asp Asn Gly Leu Val Glu Arg 405 410 415 Phe
Val Gly Leu Cys Lys Ser Ile Val Ser Lys Lys Gly Thr Lys Asn 420 425
430 Ser Leu Cys Ile Lys Val Asn Asn Arg Asp Leu Phe Phe Val Ala Ser
435 440 445 Glu Ser Ser Tyr Asn Glu Asn Gly Ile Asn Ser Pro Lys Glu
Ile Asp 450 455 460 Asp Thr Thr Ile Thr Asn Asn Asn Tyr Lys Lys Asn
Leu Asp Glu Val465 470 475 480 Ile Leu Asp Tyr Asn Ser Asp Ala Ile
Pro Asn Leu Ser Ser Arg Leu 485 490 495 Leu Asn Thr Thr Ala Gln Asn
Asp Ser Tyr Val Pro Lys Tyr Asp Ser 500 505 510 Asn Gly Thr Ser Glu
Ile Lys Glu Tyr Thr Val Asp Lys Leu Asn Val 515 520 525 Phe Phe Tyr
Leu Tyr Ala Gln Lys Ala Pro Glu Gly Glu Ser Ala Ile 530 535 540 Ser
Leu Thr Ser Ser Val Asn Thr Ala Leu Leu Asp Ala Ser Lys Val545 550
555 560 Tyr Thr Phe Phe Ser Ser Asp Phe Ile Asn Thr Val Asn Lys Pro
Val 565 570 575 Gln Ala Ala Leu Phe Ile Ser Trp Ile Gln Gln Val Ile
Asn Asp Phe 580 585 590 Thr Thr Glu Ala Thr Gln Lys Ser Thr Ile Asp
Lys Ile Ala Asp Ile 595 600 605 Ser Leu Ile Val Pro Tyr Val Gly Leu
Ala Leu Asn Ile Gly Asn Glu 610 615 620 Val Gln Lys Gly Asn Phe Lys
Glu Ala Ile Glu Leu Leu Gly Ala Gly625 630 635 640 Ile Leu Leu Glu
Phe Val Pro Glu Leu Leu Ile Pro Thr Ile Leu Val 645 650 655 Phe Thr
Ile Lys Ser Phe Ile Asn Ser Asp Asp Ser Lys Asn Lys Ile 660 665 670
Ile Lys Ala Ile Asn Asn Ala Leu Arg Glu Arg Glu Leu Lys Trp Lys 675
680 685 Glu Val Tyr Ser Trp Ile Val Ser Asn Trp Leu Thr Arg Ile Asn
Thr 690 695 700 Gln Phe Asn Lys Arg Lys Glu Gln Met Tyr Gln Ala Leu
Gln Asn Gln705 710 715 720 Val Asp Gly Ile Lys Lys Ile Ile Glu Tyr
Lys Tyr Asn Asn Tyr Thr 725 730 735 Leu Asp Glu Lys Asn Arg Leu Arg
Ala Glu Tyr Asn Ile Tyr Ser Ile 740 745 750 Lys Glu Glu Leu Asn Lys
Lys Val Ser Leu Ala Met Gln Asn Ile Asp 755 760 765 Arg Phe Leu Thr
Glu Ser Ser Ile Ser Tyr Leu Met Lys Leu Ile Asn 770 775 780 Glu Ala
Lys Ile Asn Lys Leu Ser Glu Tyr Asp Lys Arg Val Asn Gln785 790 795
800 Tyr Leu Leu Asn Tyr Ile Leu Glu Asn Ser Ser Thr Leu Gly Thr Ser
805 810 815 Ser Val Pro Glu Leu Asn Asn Leu Val Ser Asn Thr Leu Asn
Asn Ser 820 825 830 Ile Pro Phe Glu Leu Ser Glu Tyr Thr Asn Asp Lys
Ile Leu Ile His 835 840 845 Ile Leu Ile Arg Phe Tyr Lys Arg Ile Ile
Asp Ser Ser Ile Leu Asn 850 855 860 Met Lys Tyr Glu Asn Asn Arg Phe
Ile Asp Ser Ser Gly Tyr Gly Ser865 870 875 880 Asn Ile Ser Ile Asn
Gly Asp Ile Tyr Ile Tyr Ser Thr Asn Arg Asn 885 890 895 Gln Phe Gly
Ile Tyr Ser Ser Arg Leu Ser Glu Val Asn Ile Thr Gln 900 905 910 Asn
Asn Thr Ile Ile Tyr Asn Ser Arg Tyr Gln Asn Phe Ser Val Ser 915 920
925 Phe Trp Val Arg Ile Pro Lys Tyr Asn Asn Leu Lys Asn Leu Asn Asn
930 935 940 Glu Tyr Thr Ile Ile Asn Cys Met Arg Asn Asn Asn Ser Gly
Trp Lys945 950 955 960 Ile Ser Leu Asn Tyr Asn Asn Ile Ile Trp Thr
Leu Gln Asp Thr Thr 965 970 975 Gly Asn Asn Gln Lys Leu Val Phe Asn
Tyr Thr Gln Met Ile Asp Ile 980 985 990 Ser Asp Tyr Ile Asn Lys Trp
Thr Phe Val Thr Ile Thr Asn Asn Arg 995 1000 1005 Leu Gly His Ser
Lys Leu Tyr Ile Asn Gly Asn Leu Thr Asp Gln Lys 1010 1015 1020 Ser
Ile Leu Asn Leu Gly Asn Ile His Val Asp Asp Asn Ile Leu Phe1025
1030 1035 1040Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile
Arg Tyr Phe 1045 1050 1055 Lys Ile Phe Asn Met Glu Leu Asp Lys Thr
Glu Ile Glu Thr Leu Tyr 1060 1065 1070 His Ser Glu Pro Asp Ser Thr
Ile Leu Lys Asp Phe Trp Gly Asn Tyr 1075 1080 1085 Leu Leu Tyr Asn
Lys Lys Tyr Tyr Leu Leu Asn Leu Leu Lys Pro Asn 1090 1095 1100 Met
Ser Val Thr Lys Asn Ser Asp Ile Leu Asn Ile Asn Arg Gln Arg1105
1110 1115 1120Gly Ile Tyr Ser Lys Thr Asn Ile Phe Ser Asn Ala Arg
Leu Tyr Thr 1125 1130 1135 Gly Val Glu Val Ile Ile Arg Lys Val Gly
Ser Thr Asp Thr Ser Asn 1140 1145 1150 Thr Asp Asn Phe Val Arg Lys
Asn Asp Thr Val Tyr Ile Asn Val Val 1155 1160 1165 Asp Gly Asn Ser
Glu Tyr Gln Leu Tyr Ala Asp Val Ser Thr Ser Ala 1170 1175 1180 Val
Glu Lys Thr Ile Lys Leu Arg Arg Ile Ser Asn Ser Asn Tyr Asn1185
1190 1195 1200Ser Asn Gln Met Ile Ile Met Asp Ser Ile Gly Asp Asn
Cys Thr Met 1205 1210 1215 Asn Phe Lys Thr Asn Asn Gly Asn Asp Ile
Gly Leu Leu Gly Phe His 1220 1225 1230 Leu Asn Asn Leu Val Ala Ser
Ser Trp Tyr Tyr Lys Asn Ile Arg Asn 1235 1240 1245 Asn Thr Arg Asn
Asn Gly Cys Phe Trp Ser Phe Ile Ser Lys Glu His 1250 1255 1260 Gly
Trp Gln Glu1265 241251PRTClostridium butyricum 1 24Met Pro Thr Ile
Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg1 5 10 15 Thr Ile
Leu Tyr Ile Lys Pro Gly Gly Cys Gln Gln Phe Tyr Lys Ser 20 25 30
Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35
40 45 Gly Thr Ile Pro Gln Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn
Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp
Gln Glu Lys65 70 75 80 Asp Lys Phe Leu Lys Ile Val Thr Lys Ile Phe
Asn Arg Ile Asn Asp 85 90 95 Asn Leu Ser Gly Arg Ile Leu Leu Glu
Glu Leu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr
Pro Asp Gly Asp Phe Ile Ile Asn Asp 115 120 125 Ala Ser Ala Val Pro
Ile Gln Phe Ser Asn Gly Ser Gln Ser Ile Leu 130 135 140 Leu Pro Asn
Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr145 150 155 160
Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165
170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser
Phe 180 185 190 Arg Phe Lys Asp Asn Ser Met Asn Glu Phe Ile Gln Asp
Pro Ala Leu 195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His
Gly Leu Tyr Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile
Thr Gln Lys Gln Asn Pro Leu225 230 235 240 Ile Thr Asn Ile Arg Gly
Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu
Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn
Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285
Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290
295 300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val
Asn305 310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr
Ser Phe Thr Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys
Cys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu
Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly
Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn
Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr385 390 395 400 Gly
Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410
415 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly
420 425 430 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp
Asn Ile 435 440
445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr
450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu
Ser Ala465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr
Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly
Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn Val
Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn
Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu
Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550 555 560
Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Gly Trp Ile 565
570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser
Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr
Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly
Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu
Leu Glu Phe Glu Pro Glu Leu625 630 635 640 Leu Ile Pro Thr Ile Leu
Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys
Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg
Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685
Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690
695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile
Glu705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn
Glu Leu Thr Asn 725 730 735 Lys Tyr Asp Ile Glu Gln Ile Glu Asn Glu
Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp Arg
Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu Ile
Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn
Val Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His785 790 795 800 Gly
Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Ile 805 810
815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp
820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg
Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp
Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile
Asn Gly Asp Val Tyr Lys865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln
Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile Ser
Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe
Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys
Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935
940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile
Ile945 950 955 960 Trp Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys
Leu Ala Phe Asn 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr
Ile Asn Lys Trp Ile Phe 980 985 990 Val Thr Ile Thr Asn Asp Arg Leu
Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp
Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 Val Ser
Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg1025 1030
1035 1040Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu
Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro
Asn Ala Asn Ile Leu 1060 1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu
Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys
Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser 1090 1095 1100 Thr Leu
Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg1105 1110
1115 1120Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn
Ser Ser 1125 1130 1135 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln
Val Tyr Ile Asn Phe 1140 1145 1150 Val Ala Ser Lys Thr His Leu Leu
Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys
Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln
Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe1185 1190
1195 1200Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys
Ala Asp 1205 1210 1215 Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His
Met Arg Asp Asn Thr 1220 1225 1230 Asn Ser Asn Gly Phe Phe Trp Asn
Phe Ile Ser Glu Glu His Gly Trp 1235 1240 1245 Gln Glu Lys 1250
251251PRTClostridium butyricum 2 25Met Pro Lys Ile Asn Ser Phe Asn
Tyr Asn Asp Pro Val Asn Asp Arg1 5 10 15 Thr Ile Leu Tyr Ile Lys
Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met
Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr
Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60
Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys65
70 75 80 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile
Asn Asn 85 90 95 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser
Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn
Gln Phe His Ile Gly Asp 115 120 125 Ala Ser Ala Val Glu Ile Lys Phe
Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile
Met Gly Ala Glu Pro Asp Leu Phe Glu Thr145 150 155 160 Asn Ser Ser
Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly
Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185
190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu
195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr
Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys
Gln Asn Pro Leu225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile
Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile
Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala
Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val
Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala
Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310
315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr
Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln
Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu
Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile
Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu
Asn Pro Arg Ile Ile Thr Pro Ile Thr385 390 395 400 Gly Arg Gly Leu
Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val
Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435
440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn
Tyr 450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser
Glu Ser Ala465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu
Thr Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn
Gly Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn
Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu
Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu
Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550 555
560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile
565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys
Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro
Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys
Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile
Leu Leu Glu Phe Val Pro Glu Leu625 630 635 640 Leu Ile Pro Thr Ile
Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn
Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu
Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680
685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met
690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Thr Ile
Ile Glu705 710 715 720 Phe Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys
Lys Glu Leu Lys Asn 725 730 735 Asn Tyr Asp Ile Glu Gln Ile Glu Asn
Glu Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp
Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu
Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu
Asn Val Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Gln His785 790 795 800
Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Ile 805
810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr
Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys
Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn
Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn
Ile Asn Gly Glu Ile Phe Ile865 870 875 880 Tyr Pro Thr Asn Lys Asn
Gln Phe Thr Ile Phe Asn Ser Lys Pro Ser 885 890 895 Glu Val Asn Ile
Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn
Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925
Lys Ile Val Asn Ile Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930
935 940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile
Ile945 950 955 960 Trp Thr Leu Gln Asp Asn Ala Arg Ile Asn Gln Lys
Leu Val Phe Lys 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr
Ile Asn Lys Trp Ile Phe 980 985 990 Val Thr Ile Thr Asn Asp Arg Leu
Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 Gly His Leu Ile Asp
Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 Val Ser
Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg1025 1030
1035 1040Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu
Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro
Asn Thr Asn Ile Leu 1060 1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu
Leu Tyr Asp Lys Gly Tyr Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys
Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser 1090 1095 1100 Thr Leu
Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg1105 1110
1115 1120Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asp
Ser Ser 1125 1130 1135 Thr Asn Asp Arg Phe Val Arg Lys Asn Asp Gln
Val Tyr Ile Asn Tyr 1140 1145 1150 Ile Ser Asn Ser Ser Ser Tyr Ser
Leu Tyr Ala Asp Thr Asn Thr Thr 1155 1160 1165 Asp Lys Glu Lys Thr
Ile Lys Ser Ser Ser Ser Gly Asn Arg Phe Asn 1170 1175 1180 Gln Val
Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn Phe1185 1190
1195 1200Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys
Ala Asp 1205 1210 1215 Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His
Met Arg Asp His Thr 1220 1225 1230 Asn Ser Asn Gly Cys Phe Trp Asn
Phe Ile Ser Glu Glu His Gly Trp 1235 1240 1245 Gln Glu Lys 1250
265PRTHomo sapiens 26Tyr Gly Gly Phe Leu1 5 275PRTHomo sapiens
27Tyr Gly Gly Phe Met1 5 288PRTHomo sapiens 28Tyr Gly Gly Phe Met
Arg Gly Leu1 5 297PRTHomo sapiens 29Tyr Gly Gly Phe Met Arg Phe1 5
3022PRTHomo sapiens 30Tyr Gly Gly Phe Met Arg Arg Val Gly Arg Pro
Glu Trp Trp Met Asp1 5 10 15 Tyr Gln Lys Arg Tyr Gly 20
3122PRTNecturus maculosus 31Tyr Gly Gly Phe Met Arg Arg Val Gly Arg
Pro Glu Trp Trp Leu Asp1 5 10 15 Tyr Gln Lys Arg Tyr Gly 20
3222PRTBombina orientalis 32Tyr Gly Gly Phe Met Arg Arg Val Gly Arg
Pro Glu Trp Trp Gln Asp1 5 10 15 Tyr Gln Lys Arg Tyr Gly 20
3322PRTXenopus laevis 33Tyr Gly Gly Phe Met Arg Arg Val Gly Arg Pro
Glu Trp Trp Glu Asp1 5 10 15 Tyr Gln Lys Arg Tyr Gly 20
3422PRTNeoceratodus forsteri 34Tyr Gly Gly Phe Met Arg Arg Val Gly
Arg Pro Glu Trp Lys Leu Asp1 5 10 15 Asn Gln Lys Arg Tyr Gly 20
3521PRTDanio rerio 35Tyr Gly Gly Phe Met Arg Arg Val Gly Arg Pro
Asp Trp Trp Gln Glu1 5 10 15 Ser Lys Arg Tyr Gly 20 364PRTHomo
sapiens 36Tyr Pro Trp Phe1 374PRTHomo sapiens 37Tyr Pro Phe Phe1
3816PRTHomo sapiens 38Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln
Thr Pro Leu Val Thr1 5 10 15 3910PRTHomo sapiens 39Tyr Gly Gly Phe
Leu Arg Lys Tyr Pro Lys1 5 10 4031PRTHomo sapiens 40Tyr Gly Gly Phe
Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr1 5 10 15 Leu Phe
Lys Asn Ala Ile Ile Lys Asn Ala Tyr
Lys Lys Gly Glu 20 25 30 4131PRTHomo sapiens 41Tyr Gly Gly Phe Met
Ser Ser Glu Lys Ser Gln Thr Pro Leu Val Thr1 5 10 15 Leu Phe Lys
Asn Ala Ile Ile Lys Asn Ala His Lys Lys Gly Gln 20 25 30 429PRTHomo
sapiens 42Tyr Gly Gly Phe Leu Arg Lys Tyr Pro1 5 4316PRTHomo
sapiens 43Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln Thr Pro Leu
Val Thr1 5 10 15 4416PRTHomo sapiens 44Tyr Gly Gly Phe Leu Arg Arg
Ile Arg Pro Lys Leu Lys Trp Asp Asn1 5 10 15 4513PRTHomo sapiens
45Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys1 5 10
4616PRTHomo sapiens 46Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu
Lys Trp Asp Asn Gln1 5 10 15 4712PRTHomo sapiens 47Gly Gly Phe Leu
Arg Arg Ile Arg Pro Lys Leu Lys1 5 10 4817PRTXenopus laevis 48Tyr
Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Arg Trp Asp Asn1 5 10
15 Gln4917PRTXenopus laevis 49Tyr Gly Gly Phe Leu Arg Arg Ile Arg
Pro Arg Leu Arg Trp Asp Asn1 5 10 15 Gln5017PRTProtopterus
annectens 50Tyr Gly Gly Phe Met Arg Arg Ile Arg Pro Lys Ile Arg Trp
Asp Asn1 5 10 15 Gln5117PRTDanio rerio 51Tyr Gly Gly Phe Met Arg
Arg Ile Arg Pro Lys Leu Arg Trp Asp Asn1 5 10 15 Gln5217PRTAnguilla
rostrata 52Tyr Gly Gly Phe Met Arg Arg Ile Arg Pro Lys Leu Lys Trp
Asp Ser1 5 10 15 Gln5329PRTHomo sapiens 53Tyr Gly Gly Phe Leu Arg
Arg Gln Phe Lys Val Val Thr Arg Ser Gln1 5 10 15 Glu Asp Pro Asn
Ala Tyr Ser Gly Glu Leu Phe Asp Ala 20 25 5428PRTRattus norvegicus
54Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Val Thr Arg Ser Gln1
5 10 15 Glu Asn Pro Asn Thr Tyr Ser Glu Asp Leu Asp Val 20 25
5528PRTMus musculus 55Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val
Val Thr Arg Ser Gln1 5 10 15 Glu Ser Pro Asn Thr Tyr Ser Glu Asp
Leu Asp Val 20 25 5629PRTCavia porcellus 56Tyr Gly Gly Phe Leu Arg
Arg Gln Phe Lys Val Val Thr Arg Ser Gln1 5 10 15 Glu Asp Pro Asn
Ala Tyr Ser Glu Glu Phe Phe Asp Val 20 25 5729PRTSus scrofa 57Tyr
Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Val Thr Arg Ser Gln1 5 10
15 Glu Asp Pro Asn Ala Tyr Tyr Glu Glu Leu Phe Asp Val 20 25
5829PRTCanis familiaris 58Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys
Val Val Thr Arg Ser Gln1 5 10 15 Glu Asp Pro Asn Ala Tyr Ser Gly
Glu Leu Leu Asp Gly 20 25 5929PRTBos taurus 59Tyr Gly Gly Phe Leu
Arg Arg Gln Phe Lys Val Val Thr Arg Ser Gln1 5 10 15 Glu Asp Pro
Ser Ala Tyr Tyr Glu Glu Leu Phe Asp Val 20 25 6029PRTBufo marinus
60Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Thr Thr Arg Ser Glu1
5 10 15 Glu Asp Pro Ser Thr Phe Ser Gly Glu Leu Ser Asn Leu 20 25
6129PRTBombina orientalis 61Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys
Val Thr Thr Arg Ser Glu1 5 10 15 Glu Glu Pro Gly Ser Phe Ser Gly
Glu Ile Ser Asn Leu 20 25 6229PRTXenopus laevis 62Tyr Gly Gly Phe
Leu Arg Arg Gln Phe Lys Val Asn Ala Arg Ser Glu1 5 10 15 Glu Asp
Pro Thr Met Phe Ser Asp Glu Leu Ser Tyr Leu 20 25 6329PRTXenopus
laevis 63Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Asn Ala Arg
Ser Glu1 5 10 15 Glu Asp Pro Thr Met Phe Ser Gly Glu Leu Ser Tyr
Leu 20 25 6429PRTPolypterus senegalus 64Tyr Gly Gly Phe Leu Arg Arg
His Phe Lys Ile Ser Val Arg Ser Asp1 5 10 15 Glu Glu Pro Ser Ser
Tyr Ser Asp Glu Val Leu Glu Leu 20 25 6527PRTDanio rerio 65Tyr Gly
Gly Phe Leu Arg Arg His Phe Lys Ile Ser Val Arg Ser Asp1 5 10 15
Glu Glu Pro Ser Ser Tyr Glu Asp Tyr Ala Leu 20 25 6627PRTAnguilla
rostrata 66Tyr Gly Gly Phe Leu Arg Arg His Phe Lys Ile Ser Val Arg
Ser Asp1 5 10 15 Glu Glu Pro Gly Ser Tyr Asp Val Ile Gly Leu 20 25
6729PRTNeoceratodus forsteri 67Tyr Gly Gly Phe Leu Arg Arg His Phe
Lys Ile Thr Val Arg Ser Asp1 5 10 15 Glu Asp Pro Ser Pro Tyr Leu
Asp Glu Phe Ser Asp Leu 20 25 6827PRTOncorhynchus masou 68Tyr Gly
Gly Phe Leu Arg Arg His Tyr Lys Leu Ser Val Arg Ser Asp1 5 10 15
Glu Glu Pro Ser Ser Tyr Asp Asp Phe Gly Leu 20 25 6913PRTHomo
sapiens 69Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Val Thr1 5 10
7013PRTBufo marinus 70Tyr Gly Gly Phe Leu Arg Arg Gln Phe Lys Val
Thr Thr1 5 10 7113PRTXenopus laevis 71Tyr Gly Gly Phe Leu Arg Arg
Gln Phe Lys Val Asn Ala1 5 10 7213PRTPolypterus senegalus 72Tyr Gly
Gly Phe Leu Arg Arg His Phe Lys Ile Ser Val1 5 10
7313PRTNeoceratodus forsteri 73Tyr Gly Gly Phe Leu Arg Arg His Phe
Lys Ile Thr Val1 5 10 7413PRTOncorhynchus masou 74Tyr Gly Gly Phe
Leu Arg Arg His Tyr Lys Leu Ser Val1 5 10 7517PRTHomo sapiens 75Phe
Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Arg Lys Asn1 5 10
15 Gln7617PRTHomo sapiens 76Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser
Ala Arg Lys Leu Ala Asn1 5 10 15 Gln7717PRTHomo sapiens 77Phe Gly
Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Tyr Ala Asn1 5 10 15
Gln7811PRTHomo sapiens 78Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser
Ala1 5 10 7911PRTHomo sapiens 79Phe Gly Gly Phe Thr Gly Ala Arg Lys
Tyr Ala1 5 10 8011PRTHomo sapiens 80Phe Gly Gly Phe Thr Gly Ala Arg
Lys Ser Tyr1 5 10 8113PRTHomo sapiens 81Phe Gly Gly Phe Thr Gly Ala
Arg Lys Ser Ala Arg Lys1 5 10 8230PRTHomo sapiens 82Met Pro Arg Val
Arg Ser Leu Phe Gln Glu Gln Glu Glu Pro Glu Pro1 5 10 15 Gly Met
Glu Glu Ala Gly Glu Met Glu Gln Lys Gln Leu Gln 20 25 30
8317PRTHomo sapiens 83Phe Ser Glu Phe Met Arg Gln Tyr Leu Val Leu
Ser Met Gln Ser Ser1 5 10 15 Gln 848PRTHomo sapiens 84Thr Leu His
Gln Asn Gly Asn Val1 5 8510PRTHomo sapiens 85Leu Val Val Tyr Pro
Trp Thr Gln Arg Phe1 5 10 869PRTHomo sapiens 86Val Val Tyr Pro Trp
Thr Gln Arg Phe1 5 878PRTHomo sapiens 87Val Tyr Pro Trp Thr Gln Arg
Phe1 5 887PRTHomo sapiens 88Tyr Pro Trp Thr Gln Arg Phe1 5
899PRTHomo sapiens 89Leu Val Val Tyr Pro Trp Thr Gln Arg1 5
908PRTHomo sapiens 90Leu Val Val Tyr Pro Trp Thr Gln1 5 917PRTHomo
sapiens 91Val Val Tyr Pro Trp Thr Gln1 5 927PRTHomo sapiens 92Leu
Val Val Tyr Pro Trp Thr1 5 936PRTHomo sapiens 93Leu Val Val Tyr Pro
Trp1 5 9430PRTHomo sapiens 94Gly Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro His Ala Val1 5 10 15 Gly Asn His Arg Ser Phe Ser
Asp Lys Asn Gly Leu Thr Ser 20 25 30 9559PRTHomo sapiens 95Glu Leu
Arg Pro Glu Asp Asp Met Lys Pro Gly Ser Phe Asp Arg Ser1 5 10 15
Ile Pro Glu Asn Asn Ile Met Arg Thr Ile Ile Glu Phe Leu Ser Phe 20
25 30 Leu His Leu Lys Glu Ala Gly Ala Leu Asp Arg Leu Leu Asp Leu
Pro 35 40 45 Ala Ala Ala Ser Ser Glu Asp Ile Glu Arg Ser 50 55
9611PRTHomo sapiens 96Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met1
5 10 9712PRTHomo sapiens 97Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu
Met Gly1 5 10 9813PRTHomo sapiens 98Arg Pro Lys Pro Gln Gln Phe Phe
Gly Leu Met Gly Lys1 5 10 9914PRTHomo sapiens 99Arg Pro Lys Pro Gln
Gln Phe Phe Gly Leu Met Gly Lys Arg1 5 10 10036PRTHomo sapiens
100Asp Ala Asp Ser Ser Ile Glu Lys Gln Val Ala Leu Leu Lys Ala Leu1
5 10 15 Tyr Gly His Gly Gln Ile Ser His Lys Arg His Lys Thr Asp Ser
Phe 20 25 30 Val Gly Leu Met 35 10119PRTHomo sapiens 101Gly His Gly
Gln Ile Ser His Lys Arg His Lys Thr Asp Ser Phe Val1 5 10 15 Gly
Leu Met10210PRTHomo sapiens 102His Lys Thr Asp Ser Phe Val Gly Leu
Met1 5 10 10310PRTRattus norvegicus 103Asp Met His Asp Phe Phe Val
Gly Leu Met1 5 10 10410PRTHomo sapiens 104Ser Arg Thr Arg Gln Phe
Tyr Gly Leu Met1 5 10 10510PRTHomo sapiens 105Gly Lys Ala Ser Gln
Phe Phe Gly Leu Met1 5 10 10614PRTHomo sapiens 106Lys Lys Ala Tyr
Gln Leu Glu His Thr Phe Gln Gly Leu Leu1 5 10 10714PRTHomo sapiens
107Val Gly Ala Tyr Gln Leu Glu His Thr Phe Gln Gly Leu Leu1 5 10
10836PRTHomo sapiens 108Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp
Ala Pro Ala Glu Asp1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg
His Tyr Ile Asn Leu Ile Thr 20 25 30 Arg Gln Arg Tyr 35
10936PRTHomo sapiens 109Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp
Ala Ser Pro Glu Glu1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg
His Tyr Leu Asn Leu Val Thr 20 25 30 Arg Gln Arg Tyr 35
11033PRTHomo sapiens 110Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro
Glu Gln Met Ala Gln1 5 10 15 Tyr Ala Ala Asp Leu Arg Arg Tyr Ile
Asn Met Leu Thr Arg Pro Arg 20 25 30 Tyr11136PRTHomo sapiens 111Ala
Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln1 5 10
15 Met Ala Gln Tyr Ala Ala Asp Leu Arg Arg Tyr Ile Asn Met Leu Thr
20 25 30 Arg Pro Arg Tyr 35 11220PRTHomo sapiens 112His Lys Glu Asp
Thr Leu Ala Phe Ser Glu Trp Gly Ser Pro His Ala1 5 10 15 Ala Val
Pro Arg 20 1138PRTArtificial SequenceConsensus sequence for
Thrombin cleavage siteVARIANT1Position 1 is S, T, N, Q, H, K, R, F,
W, Y, G, P, A, V, L, I or MVARIANT2, 6, 7, 8Position 2, 6, 7, and
8, are independently any amino acidVARIANT3Position 3 is F, S, T,
an amidic amino acid like N or Q, or an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and MVARIANT4Position 4 is K or
RVARIANT5Position 5 is S, T, a positive amino acid like H, K, and
R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and M 113Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 1146PRTArtificial
SequenceThrombin cleavage site 114Leu Val Pro Arg Gly Ser1 5
1156PRTArtificial SequenceThrombin cleavage site 115Leu Val Pro Lys
Gly Ser1 5 1166PRTArtificial SequenceThrombin cleavage site 116Phe
Ile Pro Arg Thr Phe1 5 1176PRTArtificial SequenceThrombin cleavage
site 117Val Leu Pro Arg Ser Phe1 5 1186PRTArtificial
SequenceThrombin cleavage site 118Ile Val Pro Arg Ser Phe1 5
1196PRTArtificial SequenceThrombin cleavage site 119Ile Val Pro Arg
Gly Tyr1 5 1206PRTArtificial SequenceThrombin cleavage site 120Val
Val Pro Arg Gly Val1 5 1216PRTArtificial SequenceThrombin cleavage
site 121Val Leu Pro Arg Leu Ile1 5 1226PRTArtificial
SequenceThrombin cleavage site 122Val Met Pro Arg Ser Leu1 5
1236PRTArtificial SequenceThrombin cleavage site 123Met Phe Pro Arg
Ser Leu1 5 1248PRTArtificial SequenceConsensus sequence for
Coagulation Factor VIIa cleavage siteVARIANT1Position 1 is an
acidic amino acid like D and E, an amidic amino acid like N and Q,
a basic amino acid like K and R, or an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and MVARIANT2Position 2 is Q, S, T, an
aromatic hydrophobic amino acid like F, W and Y, or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and
MVARIANT3Position 3 is Q, S, T, or an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and MVARIANT4Position 4 is K or
RVARIANT5, 6, 7 8Position 5, 6, 7, and 8 are independently any
amino acid 124Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 1258PRTArtificial
SequenceCoagulation Factor VIIa cleavage site 125Lys Leu Thr Arg
Ala Glu Thr Val1 5 1268PRTArtificial SequenceCoagulation Factor
VIIa cleavage site 126Asp Phe Thr Arg Val Val Gly Gly1 5
1278PRTArtificial SequenceCoagulation Factor VIIa cleavage site
127Leu Ser Pro Arg Thr Phe His Pro1 5 1288PRTArtificial
SequenceCoagulation Factor VIIa cleavage site 128Leu Ile Gln Arg
Asn Leu Ser Pro1 5 1298PRTArtificial SequenceCoagulation Factor
VIIa cleavage site 129Met Ala Thr Arg Lys Met His Asp1 5
1308PRTArtificial SequenceCoagulation Factor VIIa cleavage site
130Leu Gly Ile Arg Ser Phe Arg Asn1 5 1318PRTArtificial
SequenceCoagulation Factor VIIa cleavage site 131Pro Gln Gly Arg
Ile Val Gly Gly1 5 1328PRTArtificial SequenceCoagulation Factor
VIIa cleavage site 132Asn Leu Thr Arg Ile Val Gly Gly1 5
1338PRTArtificial SequenceCoagulation Factor VIIa cleavage site
133Gln Val Val Arg Ile Val Gly Gly1 5 1348PRTArtificial
SequenceConsenus sequence for Coagulation Factor IXa cleavage
siteVARIANT1Position 1 is an acidic amino acid like D and E, an
amidic amino acid like N and Q, or an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and MVARIANT2Position 2 is an acidic
amino acid like D and E, an amidic amino acid like N and Q, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
MVARIANT3Position 3 is, S, T, an aromatic hydrophobic amino acid
like F, W and Y, or an aliphatic hydrophobic amino acid like, G, P,
A, V, L, I, and MVARIANT4Position 4 is K or RVARIANT5, 6, 7,
8Positions 5, 6, 7, and 8 are independently any amino acid 134Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 1358PRTArtificial
SequenceCoagulation Factor IXa cleavage site 135Pro Gln Gly Arg Ile
Val Gly Gly1 5 1368PRTArtificial SequenceCoagulation Factor IXa
cleavage site 136Pro Gln Leu Arg Met Lys Asn Asn1 5
1378PRTArtificial SequenceCoagulation Factor IXa cleavage site
137Asn Leu Thr Arg Ile Val Gly Gly1 5 1388PRTArtificial
SequenceCoagulation Factor IXa cleavage site 138Gln Val Val Arg Ile
Val Gly Gly1 5 1398PRTArtificial SequenceConsensus sequence for
Coagulation Factor Xa cleavage siteVARIANT1, 6, 7, 8Positions 1, 6,
7, and 8 are independantly any amino acidVARIANT2Position 2 is G,
A, S, an acidic amino acid like D and E, an amidic amino acid like
Q and N, or an aromatic hydrophobic amino acid like F, W and
YVARIANT3Position 3 is an aromatic hydrophobic amino acid like F, W
and Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and MVARIANT4Position 4 is K or
RVARIANT5Position 5 is an amidic amino acid like N and Q, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M 139Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 1404PRTArtificial SequenceCoagulation Factor Xa cleavage
site 140Ile Asp Gly Arg1 1414PRTArtificial SequenceCoagulation
Factor Xa cleavage site 141Ile Glu Gly Arg1 1428PRTArtificial
SequenceCoagulation Factor Xa cleavage site 142Ile Asp Gly Arg Ser
Val Gly Gly1 5 1438PRTArtificial SequenceCoagulation Factor Xa
cleavage site 143Ile Asp Gly Arg Thr Val Gly Gly1 5
1448PRTArtificial SequenceCoagulation Factor Xa cleavage site
144Ile Asp Gly Arg Ile Val Gly Gly1 5 1458PRTArtificial
SequenceCoagulation Factor Xa cleavage site 145Ile Glu Gly Arg Ser
Val Gly Gly1 5 1468PRTArtificial SequenceCoagulation Factor Xa
cleavage site 146Ile Glu Gly Arg Thr Val Gly Gly1 5
1478PRTArtificial SequenceCoagulation Factor Xa cleavage site
147Ile Glu Gly Arg Ile Val Gly Gly1 5 1488PRTArtificial
SequenceCoagulation Factor Xa cleavage site 148Pro Gln Gly Arg Ile
Val Gly Gly1 5 1498PRTArtificial SequenceCoagulation Factor Xa
cleavage site 149Ile Glu Gly Arg Thr Ser Glu Asp1 5
1508PRTArtificial SequenceCoagulation Factor Xa cleavage site
150Ile Glu Gly Arg Ile Val Glu Gly1 5 1518PRTArtificial
SequenceCoagulation Factor Xa cleavage site 151Ile Asp Gly Arg Ile
Val Glu Gly1 5 1528PRTArtificial SequenceCoagulation Factor Xa
cleavage site 152Phe Asn Pro Arg Thr Phe Gly Ser1 5
1538PRTArtificial SequenceCoagulation Factor Xa cleavage site
153Phe Asp Glu Arg Thr Phe Gly Leu1 5 1548PRTArtificial
SequenceCoagulation Factor Xa cleavage site 154Ile Asp Glu Arg Ile
Val Gly Gly1 5 1558PRTArtificial SequenceCoagulation Factor Xa
cleavage site 155Phe Asn Glu Lys Thr Phe Gly Leu1 5
1568PRTArtificial SequenceConsensus sequence for Coagulation Factor
XIa cleavage siteVARIANT1Position 1 is an acidic amino acid like D
or E, a basic amino acid like K and R, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and MVARIANT2Position 2 is D, E,
Q, N, K, R, F, W, Y, G, P, A, V, L, I, or MVARIANT3Position 3 is H,
C, S, T, F, W, Y, G, P, A, V, L, I, or MVARIANT4Position 4 is K or
RVARIANT5, 8Positions 5 and 6 are independantly H, an uncharged
amino acid like C, S, and T, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and MVARIANT(6)...(6)Position 6 is an
acidic amino acid like D and E, or an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and MVARIANT(7)...(7)Position 7 is Q,
N, C, S, T, F, W, Y, G, P, A, V, L, I, or M 156Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 1578PRTArtificial SequenceCoagulation Factor XIa
cleavage site 157Ala Phe Trp Lys Thr Asp Ala Ser1 5
1588PRTArtificial SequenceCoagulation Factor XIa cleavage site
158Lys Leu Thr Arg Ala Glu Thr Val1 5 1598PRTArtificial
SequenceCoagulation Factor XIa cleavage site 159Lys Leu Thr Arg Ala
Glu Thr Ile1 5 1608PRTArtificial SequenceCoagulation Factor XIa
cleavage site 160Asp Phe Thr Arg Val Val Gly Gly1 5
1618PRTArtificial SequenceCoagulation Factor XIa cleavage site
161Glu Phe Ser Arg Val Val Gly Gly1 5 1628PRTArtificial
SequenceCoagulation Factor XIa cleavage site 162Lys Leu Thr Arg Ala
Glu Thr Val1 5 1638PRTArtificial SequenceCoagulation Factor XIa
cleavage site 163Asp Phe Thr Arg Val Val Gly Gly1 5
1648PRTArtificial SequenceCoagulation Factor XIa cleavage site
164Ile Lys Pro Arg Ile Val Gly Gly1 5 1658PRTArtificial
SequenceCoagulation Factor XIa cleavage site 165Asp Leu His Arg His
Ile Phe Trp1 5 1668PRTArtificial SequenceCoagulation Factor XIa
cleavage site 166Lys Gln Leu Arg Val Val Asn Gly1 5
1678PRTArtificial SequenceConsensus sequence for Coagulation Factor
XIIa cleavage siteVARIANT1, 6Positions 1 and 6 are independantly an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and MVARIANT2Position 2 is an
acidic amino acid like D and E, a basic amino acid like K and R, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and MVARIANT3Position 3 is a
basic amino acid like K and R, an uncharged amino acid like C, S,
and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,
I, and MVARIANT4Position 4 is K or RVARIANT5, 7, 8Positions 5, 7
and 8 are independantly an aliphatic hydrophobic amino acid like,
G, P, A, V, L, I, and M 167Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
1688PRTArtificial SequenceCoagulation Factor XIIa cleavage site
168Pro Gln Gly Arg Ile Val Gly Gly1 5 1698PRTArtificial
SequenceCoagulation Factor XIIa cleavage site 169Ile Lys Pro Arg
Ile Val Gly Gly1 5 1708PRTArtificial SequenceCoagulation Factor
XIIa cleavage site 170Ser Met Thr Arg Val Val Gly Gly1 5
1718PRTArtificial SequenceCoagulation Factor XIIa cleavage site
171Thr Ser Thr Arg Ile Val Gly Gly1 5 1728PRTArtificial
SequenceCoagulation Factor XIIa cleavage site 172Pro Met Lys Arg
Leu Thr Leu Gly1 5 1738PRTArtificial SequenceConsensus sequence for
Kallikrein 1 cleavage siteVARIANT1Position 1 is D, E, Q, N, C, S,
T, G, P, A, V, L, I or MVARIANT2, 3, 6, 7, 8Positions 2, 3, 6, 7
and 8 are independently any amino acidVARIANT4Position 4 is a
positive amino acid like H, K, and R, a large non-polar amino acid
like F, I, L, M and V, or an aromatic hydrophobic amino acid like
F, W and YVARIANT5Position 5 is S, K, or R 173Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 1748PRTArtificial SequenceKallikrein 1 cleavage site
174Ser Met Thr Arg Val Val Gly Gly1 5 1758PRTArtificial
SequenceKallikrein 1 cleavage site 175Ser Pro Phe Arg Ser Ser Asp
Ile1 5 1768PRTArtificial SequenceKallikrein 1 cleavage site 176Ser
Leu Met Lys Arg Pro Pro Gly1 5 1778PRTArtificial SequenceKallikrein
1 cleavage site 177Tyr Asp Trp Arg Thr Pro Tyr Leu1 5
1788PRTArtificial SequenceKallikrein 1 cleavage site 178Ser Pro Phe
Arg Ser Val Gln Val1 5 1798PRTArtificial SequenceKallikrein 1
cleavage site 179Ser Pro Phe Arg Thr Pro Tyr Leu1 5
1808PRTArtificial SequenceKallikrein 1 cleavage site 180Thr Phe His
Lys Ala Glu Tyr Arg1 5 1818PRTArtificial SequenceKallikrein 1
cleavage site 181Pro Arg Phe Lys Ile Ile Gly Gly1 5
1828PRTArtificial SequenceKallikrein 1 cleavage site 182Ile Ser Leu
Met Lys Arg Pro Pro1 5 1838PRTArtificial SequenceKallikrein 1
cleavage site 183Leu Glu Ala Arg Ser Ala Tyr His1 5
1848PRTArtificial SequenceKallikrein 1 cleavage site 184Glu Ala Lys
Arg Ser Tyr His Ser1 5 1858PRTArtificial SequenceKallikrein 1
cleavage site 185Pro Asn Arg Trp Ser Thr Gly Ala1 5
1868PRTArtificial SequenceKallikrein 1 cleavage site 186Glu Ala Phe
Tyr Ser Gln Phe Gly1 5 1878PRTArtificial SequenceKallikrein 1
cleavage site 187Asn Ala Ala Arg Ser Thr Gly Ala1 5
1888PRTArtificial SequenceKallikrein 1 cleavage site 188Ser Ser Glu
Trp Ser Met Pro Tyr1 5 1898PRTArtificial SequenceKallikrein 1
cleavage site 189Gly Thr Leu Phe Arg Ser Gly Asn1 5
1908PRTArtificial SequenceKallikrein 1 cleavage site 190Ala Arg Leu
Tyr Ser Arg Gly Ala1 5 1918PRTArtificial SequenceKallikrein 1
cleavage site 191Glu Ala Ser Arg Ser Ala Thr Leu1 5
1928PRTArtificial SequenceKallikrein 1 cleavage site 192Glu Ala Ser
Tyr Arg Arg Lys Gln1 5 1938PRTArtificial SequenceKallikrein 1
cleavage site 193Thr Thr Phe Tyr Arg Arg Gly Ala1 5
1948PRTArtificial SequenceKallikrein 1 cleavage site 194Ala Ala Trp
Tyr Arg Thr Ser Arg1 5 1958PRTArtificial SequenceKallikrein 1
cleavage site 195Ser Phe His Tyr Arg Met Val Gly1 5
1968PRTArtificial SequenceKallikrein 1 cleavage site 196Ala Ser Ser
Tyr Arg Thr Ser Arg1 5 1978PRTArtificial SequenceKallikrein 1
cleavage site 197Thr Arg Phe Tyr Ser Arg Gly Arg1 5
1988PRTArtificial SequenceKallikrein 1 cleavage site 198Ile Lys Phe
Phe Ser Ala Gln Thr1 5 1998PRTArtificial SequenceConsensus sequence
for Protein C cleavage siteVARIANT1Position 1 is a basic amino acid
like K and R, or an aliphatic hydrophobic amino acid like, G, P, A,
V, L, I, and MVARIANT2Position 2 is an acidic amino acid like D and
E, an amidic amino acid like Q and N, a basic amino acid like K and
R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,
and MVARIANT3, 5Positions 3 and 5 is Q, N, K, R, C, S, T, G, P, A,
V, L, I or MVARIANT4Position 4 is K or RVARIANT6Position 6 is Q, N,
K, R, F, W, Y, G, P, A, V, L, I or MVARIANT(7)...(7)Position 7 is
an amidic amino acid like Q and N, a positive amino acid like H, K,
and R, an uncharged amino acid like C, S, and T, or an aromatic
hydrophobic amino acid like F, W and YVARIANT(8)...(8)Position 8 is
D, E, Q, N, K, R, C, S, T, G, P, A, V, L, I or M 199Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5 2008PRTArtificial SequenceProtein C cleavage
site 200Lys Lys Thr Arg Asn Leu Lys Lys1 5 2018PRTArtificial
SequenceProtein C cleavage site 201Leu Asp Arg Arg Gly Leu Gln Arg1
5 2028PRTArtificial SequenceProtein C cleavage site 202Met Ala Thr
Arg Lys Met His Asp1 5 2038PRTArtificial SequenceProtein C cleavage
site 203Arg Leu Lys Lys Ser Gln Phe Leu1 5 2048PRTArtificial
SequenceProtein C cleavage site 204Pro Gln Leu Arg Met Lys Asn Asn1
5 2058PRTArtificial SequenceProtein C cleavage site 205Val Asp Gln
Arg Gly Asn Gln Ile1 5 2068PRTArtificial SequenceProtein C cleavage
site 206Ile Glu Pro Arg Ser Pro Ser Gln1 5 2078PRTArtificial
SequenceProtein C cleavage site 207Lys Lys Thr Arg Ser Pro Lys Thr1
5 2088PRTArtificial SequenceProtein C cleavage site 208Leu Asp Gln
Arg Gly Val Gln Arg1 5 2098PRTArtificial SequenceProtein C cleavage
site 209Pro Asp Pro Arg Ser Lys Asn Asn1 5 2108PRTArtificial
SequenceConsensus sequence for Plasminogen cleavage siteVARIANT1,
5, 6Positions 1, 5, and 6 are independantly H, K R, C, S, T, G, P,
A, V, L, I or MVARIANT2Position 3 is N, Q, H, K, R, C, S, T, G, P,
A, V, L, I or MVARIANT3Position 3 is N, Q, C, S, T, F, W, Y, G, P,
A, V, L, I or MVARIANT4Position 4 is K or RVARIANT7Position 7 is
any amino acidVARIANT(8)...(8)Position 8 is H, F, Y, R, an
uncharged amino acid like C, S, and T, an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and M 210Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 2118PRTArtificial SequencePlasminogen cleavage site
211Gly Glu Ala Arg Gly Ser Val Ile1 5 2128PRTArtificial
SequencePlasminogen cleavage site 212Gly His Ala Arg Leu Val His
Val1 5 2138PRTArtificial SequencePlasminogen cleavage site 213Ala
Glu Phe Arg His Asp Ser Gly1 5 2148PRTArtificial
SequencePlasminogen cleavage site 214His His Gln Lys Leu Val Phe
Phe1 5 2158PRTArtificial SequencePlasminogen cleavage site 215Gly
Ser Asn Lys Gly Ala Leu Leu1 5 2168PRTArtificial
SequencePlasminogen cleavage site 216Arg Ala Gln Arg Ser Ala Gly
Ala1 5 2178PRTArtificial SequencePlasminogen cleavage site 217Ala
Phe Trp Lys Thr Asp Ala Ser1 5 2188PRTArtificial
SequencePlasminogen cleavage site 218Met Ser Met Arg Val Arg Arg
His1 5 2198PRTArtificial SequencePlasminogen cleavage site 219Arg
Gly Val Arg Arg Thr Ala Ser1 5 2208PRTArtificial
SequencePlasminogen cleavage site 220Arg Ala Ala Arg Ser Gln Cys
Thr1 5 2218PRTArtificial SequencePlasminogen cleavage site 221Pro
Gln Ser Arg Ser Val Pro Pro1 5 2228PRTArtificial
SequencePlasminogen cleavage site 222Pro Tyr Leu Lys Val Phe Asn
Pro1 5 2238PRTArtificial SequencePlasminogen cleavage site 223Leu
Ser Phe Arg Ala Arg Ala Tyr1 5 2248PRTArtificial
SequencePlasminogen cleavage site 224Pro Gln Leu Arg Arg Gly Trp
Arg1 5 2258PRTArtificial SequencePlasminogen cleavage site 225Glu
Asp Asn Arg Asp Ser Ser Met1 5 2268PRTArtificial
SequencePlasminogen cleavage site 226Leu Ser Phe Arg Ala Arg Ala
Tyr1 5 2278PRTArtificial SequencePlasminogen cleavage site 227Phe
Arg Ala Arg Ala Tyr Gly Phe1 5 2288PRTArtificial
SequencePlasminogen cleavage site 228Tyr Gly Phe Arg Gly Pro Gly
Pro1 5 2298PRTArtificial SequencePlasminogen cleavage site 229Ile
Thr Phe Arg Met Asn Val Ala1 5 2308PRTArtificial
SequencePlasminogen cleavage site 230Thr His Glu Lys Gly Arg Gln
Ser1 5 2318PRTArtificial SequencePlasminogen cleavage site 231Pro
Arg Leu Lys Ala Arg Ala Gly1 5 2328PRTArtificial
SequencePlasminogen cleavage site 232Pro Lys Ala Lys Ser His Ala
Pro1 5 2338PRTArtificial SequencePlasminogen cleavage site 233Pro
Ser His Lys Glu Gly Pro Gln1 5 2348PRTArtificial
SequencePlasminogen cleavage site 234Leu Phe Glu Lys Lys Val Tyr
Leu1 5 2358PRTArtificial SequencePlasminogen cleavage site 235Ala
Asp Gly Lys Lys Pro Ser Ser1 5 2368PRTArtificial
SequencePlasminogen cleavage site 236Pro Arg Phe Lys Ile Ile Gly
Gly1 5 2378PRTArtificial SequencePlasminogen cleavage site 237Pro
Gln Phe Arg Ile Lys Gly Gly1 5 2388PRTArtificial
SequencePlasminogen cleavage site 238Pro Arg Cys Arg His Arg Pro
His1 5 2398PRTArtificial SequencePlasminogen cleavage site 239Lys
Gly Tyr Arg Ser Gln Arg Gly1 5 2408PRTArtificial
SequencePlasminogen cleavage site 240Asp Val Ala Gln Phe Val Leu
Thr1 5 2418PRTArtificial SequenceConsensus sequence for MMP-2
cleavage siteVARIANT1, 3, 4, 6, 7, 8Positions 1, 3, 4, 6, 7, 8 are
independantly any amino acidVARIANT2Position 2 is P, A, V, L, or
IVARIANT5Position 5 is V, L, I, F, or Q 241Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 2428PRTArtificial SequenceMMP-2 cleavage site 242Gln Pro
Val Ser Val Lys Val Gly1 5 2438PRTArtificial SequenceMMP-2 cleavage
site 243Arg Gly Val Gly Ile Lys Ser Thr1 5 2448PRTArtificial
SequenceMMP-2 cleavage site 244Phe Val Asp Cys Leu Ile Glu Gln1 5
2458PRTArtificial SequenceMMP-2 cleavage site 245Val Pro Ala Gly
Asn Trp Val Leu1 5 2468PRTArtificial SequenceMMP-2 cleavage site
246Tyr His Ala Asp Ile Tyr Asp Lys1 5 2478PRTArtificial
SequenceMMP-2 cleavage site 247Arg Ala Cys Arg Leu Ala Lys Ala1 5
2488PRTArtificial SequenceMMP-2 cleavage site 248Gln Gly Ala Tyr
Gln Glu Ala Phe1 5 2498PRTArtificial SequenceMMP-2 cleavage site
249Asp Val Leu Ser Leu Leu Glu Lys1 5 2508PRTArtificial
SequenceMMP-2 cleavage site 250Thr Leu Asp Asp Leu Ile Met Ala1 5
2518PRTArtificial SequenceMMP-2 cleavage site 251His Ile Ser Ser
Leu Ile Lys Leu1 5 2528PRTArtificial SequenceMMP-2 cleavage site
252Asp Pro Asn Asn Leu Leu Asn Asp1 5 2538PRTArtificial
SequenceMMP-2 cleavage site 253Pro Val Gln Pro Gln Gln Ser Pro1 5
2548PRTArtificial SequenceMMP-2 cleavage site 254Lys Pro Lys Thr
Ile Thr Gly Pro1 5 2558PRTArtificial SequenceMMP-2 cleavage site
255Val Val His Pro Leu Val Leu Leu1 5 2568PRTArtificial
SequenceMMP-2
cleavage site 256His Pro Leu Val Leu Leu Ser Val1 5
2578PRTArtificial SequenceMMP-2 cleavage site 257Ala Val Ala Leu
Leu Ile Gly Pro1 5 2588PRTArtificial SequenceMMP-2 cleavage site
258Gln Pro Leu Gln Leu Leu Asp Ala1 5 2598PRTArtificial
SequenceMMP-2 cleavage site 259Tyr Ile Gln Gly Ile Asn Leu Val1 5
2608PRTArtificial SequenceMMP-2 cleavage site 260Leu Pro Gln Glu
Ile Lys Ala Asn1 5 2618PRTArtificial SequenceMMP-2 cleavage site
261Asn Ile Ser Asp Leu Thr Ala Ala1 5 2628PRTArtificial
SequenceMMP-2 cleavage site 262Lys Pro Arg Ala Leu Thr Ala Leu1 5
2638PRTArtificial SequenceMMP-2 cleavage site 263Ala Pro Ser Trp
Leu Leu Thr Ala1 5 2648PRTArtificial SequenceMMP-2 cleavage site
264Ala Val Arg Trp Leu Leu Thr Ala1 5 2658PRTArtificial
SequenceMMP-2 cleavage site 265Ala Val Ser Trp Leu Leu Thr Ala1 5
2668PRTArtificial SequenceMMP-2 cleavage site 266Ser Leu Arg Arg
Leu Thr Ala Ala1 5 2678PRTArtificial SequenceMMP-2 cleavage site
267Ser Leu Ser Arg Leu Thr Ala Leu1 5 2688PRTArtificial
SequenceMMP-2 cleavage site 268Arg Tyr Ser Ser Leu Thr Ala Ala1 5
2698PRTArtificial SequenceMMP-2 cleavage site 269Ser Leu Ala Tyr
Tyr Thr Ala Leu1 5 2708PRTArtificial SequenceMMP-2 cleavage site
270Ser Leu Arg Tyr Tyr Thr Ala Ala1 5 2718PRTArtificial
SequenceMMP-2 cleavage site 271Ser Pro Ala Tyr Tyr Thr Ala Leu1 5
2728PRTArtificial SequenceMMP-2 cleavage site 272Met His Lys Ala
Leu Thr Ala Ala1 5 2738PRTArtificial SequenceMMP-2 cleavage site
273Leu Arg Leu Ala Ile Thr Ala Leu1 5 2748PRTArtificial
SequenceConsensus sequence for MMP-9 cleavage siteVARIANT1Position
1 is F, D, E, N, Q, H, K, R, C, S, T, G, P, A, V, L, I or
MVARIANT2Position 2 is F, Y, S, T, D, E, N, Q, H, K, R, G, P, A, V,
L, I or MVARIANT3, 8Positions 3 and 8 are independantly F, Y, D, E,
N, Q, H, K, R, C, S, T, G, P, A, V, L, I or MVARIANT4, 6,
7Positions 4, 6 and 7 are independantly any amino
acidVARIANT5Position 5 is S, T, D, E, N, Q, H, K, R, F, W, Y, G, P,
A, V, L, I or M 274Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
2758PRTArtificial SequenceMMP-9 cleavage site 275Ile Pro Glu Asn
Phe Phe Gly Val1 5 2768PRTArtificial SequenceMMP-9 cleavage site
276Met Asp Ile Ala Ile His His Pro1 5 2778PRTArtificial
SequenceMMP-9 cleavage site 277Ser Pro Ser Arg Leu Phe Asp Gln1 5
2788PRTArtificial SequenceMMP-9 cleavage site 278Ser Glu Met Arg
Leu Glu Lys Asp1 5 2798PRTArtificial SequenceMMP-9 cleavage site
279Phe Ser Val Asn Leu Asp Val Lys1 5 2808PRTArtificial
SequenceMMP-9 cleavage site 280Arg Leu Phe Asp Gln Phe Phe Gly1 5
2818PRTArtificial SequenceMMP-9 cleavage site 281Phe Phe Gly Glu
His Leu Leu Glu1 5 2828PRTArtificial SequenceMMP-9 cleavage site
282Gly Leu Ser Glu Met Arg Leu Glu1 5 2838PRTArtificial
SequenceMMP-9 cleavage site 283Ser Pro Glu Glu Leu Lys Val Lys1 5
2848PRTArtificial SequenceMMP-9 cleavage site 284Asp Val Ile Glu
Val His Gly Lys1 5 2858PRTArtificial SequenceMMP-9 cleavage site
285Glu Val His Gly Lys His Glu Glu1 5 2868PRTArtificial
SequenceMMP-9 cleavage site 286Asp Glu His Gly Phe Ile Ser Arg1 5
2878PRTArtificial SequenceMMP-9 cleavage site 287Gly Glu His Leu
Leu Glu Ser Asp1 5 2888PRTArtificial SequenceMMP-9 cleavage site
288Phe His Arg Lys Tyr Arg Ile Pro1 5 2898PRTArtificial
SequenceMMP-9 cleavage site 289Gly Pro Arg Lys Gln Val Ser Gly1 5
2908PRTArtificial SequenceMMP-9 cleavage site 290Leu Ser Pro Phe
Tyr Leu Arg Pro1 5 2918PRTArtificial SequenceMMP-9 cleavage site
291Pro Pro Ser Phe Leu Arg Ala Pro1 5 2928PRTArtificial
SequenceMMP-9 cleavage site 292Asn Pro Leu Glu Asn Ser Gly Phe1 5
2938PRTArtificial SequenceMMP-9 cleavage site 293Val Pro Tyr Gly
Leu Gly Ser Pro1 5 2948PRTArtificial SequenceMMP-9 cleavage site
294Pro Pro Leu Lys Leu Met His Ser1 5 2958PRTArtificial
SequenceMMP-9 cleavage site 295Gly Pro Glu Gly Leu Arg Val Gly1 5
2968PRTArtificial SequenceMMP-9 cleavage site 296Phe Met Lys Gly
Leu Ser Lys Ala1 5 2978PRTArtificial SequenceMMP-9 cleavage site
297Val Val Thr Gly Val Thr Ala Val1 5 2988PRTArtificial
SequenceMMP-9 cleavage site 298Ala Ile Ile Gly Leu Met Val Gly1 5
2998PRTArtificial SequenceMMP-9 cleavage site 299Ser Asp Leu Gly
Leu Thr Gly Ile1 5 3008PRTArtificial SequenceMMP-9 cleavage site
300Val Pro Tyr Gly Leu Gly Ser Pro1 5 3018PRTArtificial
SequenceMMP-9 cleavage site 301Gly Ala Ala Gly Val Lys Gly Asp1 5
3028PRTArtificial SequenceMMP-9 cleavage site 302Gly Pro Thr Gly
Lys Gln Gly Asp1 5 3038PRTArtificial SequenceMMP-9 cleavage site
303Gly Pro Ser Gly Asp Gln Gly Ala1 5 3048PRTArtificial
SequenceMMP-9 cleavage site 304Gly Pro Ser Gly Phe Pro Phe Pro1 5
3058PRTArtificial SequenceMMP-9 cleavage site 305Gly Ala Pro Gly
Phe Pro Gly Pro1 5 3068PRTArtificial SequenceMMP-9 cleavage site
306Gly Ala Pro Gly Asn Arg Gly Phe1 5 3078PRTArtificial
SequenceMMP-9 cleavage site 307Gly Leu Arg Gly Glu Arg Gly Glu1 5
3088PRTArtificial SequenceMMP-9 cleavage site 308Gly Pro Pro Gly
Ser Gln Gly Asn1 5 3098PRTArtificial SequenceMMP-9 cleavage site
309Gly Pro Ala Gly Gln Gln Gly Ala1 5 3108PRTArtificial
SequenceMMP-9 cleavage site 310Gly Pro Pro Gly Lys Asp Gly Thr1 5
3118PRTArtificial SequenceMMP-9 cleavage site 311Gly Gln Pro Gly
Ser Pro Gly Ser1 5 3128PRTArtificial SequenceMMP-9 cleavage site
312Gly Ser Pro Gly Tyr Gln Gly Pro1 5 3138PRTArtificial
SequenceMMP-9 cleavage site 313Gly Pro Val Ser Ala Val Leu Thr1 5
3148PRTArtificial SequenceMMP-9 cleavage site 314Gly Pro Leu Gly
Met Leu Ser Gln1 5 3158PRTArtificial SequenceMMP-9 cleavage site
315Gly Pro Leu Gly Met Trp Ala Gln1 5 3168PRTArtificial
SequenceMMP-9 cleavage site 316Gly Pro Gln Gly Ile Phe Gly Gln1 5
3178PRTArtificial SequenceMMP-9 cleavage site 317Leu Pro Arg Ser
Ala Lys Glu Leu1 5 3188PRTArtificial SequenceMMP-9 cleavage site
318Asn Ser Phe Gly Leu Arg Phe Gly1 5 3198PRTArtificial
SequenceMMP-9 cleavage site 319Arg Ala Ile His Ile Asn Ala Glu1 5
3208PRTArtificial SequenceConsensus sequence for Furin cleavage
siteVARIANT1Position 1 is R, I, or AVARIANT2, 5, 6, 7, 8Positions
2, 5, 6, 7, and 8 are independantly any amino acidVARIANT3Position
3 is R, K, A, or PVARIANT(0)...(0) 320Xaa Xaa Xaa Arg Xaa Xaa Xaa
Xaa1 5 3218PRTArtificial SequenceFurin cleavage site 321Arg Pro Arg
Arg Ala Lys Arg Phe1 5 3228PRTArtificial SequenceFurin cleavage
site 322Arg Lys Lys Arg Gly Leu Tyr Ala1 5 3238PRTArtificial
SequenceFurin cleavage site 323Arg Glu Arg Arg Arg Lys Lys Arg1 5
3248PRTArtificial SequenceFurin cleavage site 324Arg Lys Lys Arg
Gly Leu Tyr Ala1 5 3258PRTArtificial SequenceFurin cleavage site
325Arg Lys Lys Arg Thr Thr Ser Ala1 5 3268PRTArtificial
SequenceFurin cleavage site 326Arg His Lys Arg Glu Thr Leu Lys1 5
3278PRTArtificial SequenceFurin cleavage site 327Arg Leu Lys Arg
Asp Val Val Thr1 5 3288PRTArtificial SequenceFurin cleavage site
328Arg Met Lys Arg Glu Asp Leu Asn1 5 3298PRTArtificial
SequenceFurin cleavage site 329Arg Ala Lys Arg Phe Ala Ser Leu1 5
3308PRTArtificial SequenceFurin cleavage site 330Arg Lys Lys Arg
Phe Val Ser Ser1 5 3318PRTArtificial SequenceFurin cleavage site
331Arg Thr Lys Arg Phe Leu Ser Tyr1 5 3328PRTArtificial
SequenceFurin cleavage site 332Arg Arg Ala Arg Ser Val Asp Gly1 5
3338PRTArtificial SequenceFurin cleavage site 333Val Phe Arg Arg
Asp Ala His Lys1 5 3348PRTArtificial SequenceFurin cleavage site
334Val Phe Arg Arg Glu Ala His Lys1 5 3358PRTArtificial
SequenceFurin cleavage site 335Arg Val Ala Arg Asp Ile Thr Met1 5
3368PRTArtificial SequenceFurin cleavage site 336Arg Ile Ser Arg
Ser Leu Pro Gln1 5 3378PRTArtificial SequenceFurin cleavage site
337Arg Ser Arg Arg Ala Ala Thr Ser1 5 3388PRTArtificial
SequenceFurin cleavage site 338Arg Ala Lys Arg Ser Pro Lys His1 5
3398PRTArtificial SequenceFurin cleavage site 339Phe Trp His Arg
Gly Val Thr Lys1 5 3408PRTArtificial SequenceFurin cleavage site
340Ala Lys Arg Arg Thr Lys Arg Asp1 5 3418PRTArtificial
SequenceFurin cleavage site 341Ala Lys Arg Arg Ala Lys Arg Asp1 5
3428PRTArtificial SequenceFurin cleavage site 342Ala Lys Gln Arg
Ala Lys Arg Asp1 5 3438PRTArtificial SequenceFurin cleavage site
343Arg Asp Val Arg Gly Phe Ala Ser1 5 3448PRTArtificial
SequenceFurin cleavage site 344Arg Lys Arg Arg Ser Val Asn Pro1 5
3458PRTArtificial SequenceFurin cleavage site 345Arg Gln Lys Arg
Phe Val Leu Ser1 5 3468PRTArtificial SequenceFurin cleavage site
346Arg Ser Lys Arg Ser Leu Ser Cys1 5 3478PRTArtificial
SequenceConsensus sequence for u-PA cleavage siteVARIANT1, 5,
8Positions 1, 5, and 8 are independantly any amino acidVARIANT2,
7Positions 2 and 7 are independantly an uncharged amino acid like
C, S, and T, an aromatic amino acid like F, W, and Y, or an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
MVARIANT3Position 3 is an amidic amino acid like N and Q, an
uncharged amino acid like C, S, and T, or an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and MVARIANT4Position 4 is K or
RVARIANT6Position 6 is a basic amino acid like K and R, an aromatic
amino acid like F, W, and Y, or an aliphatic hydrophobic amino acid
like, G, P, A, V, L, I, and M 347Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
3488PRTArtificial Sequenceu-PA cleavage site 348Gly Ser Gly Lys Ser
Ala Thr Leu1 5 3498PRTArtificial Sequenceu-PA cleavage site 349Gln
Arg Gly Arg Ser Ala Thr Leu1 5 3508PRTArtificial Sequenceu-PA
cleavage site 350Arg Gly Ser Val Ile Leu Thr Val1 5
3518PRTArtificial Sequenceu-PA cleavage site 351Pro Ser Ser Arg Arg
Arg Val Asn1 5 3528PRTArtificial Sequenceu-PA cleavage site 352Cys
Pro Gly Arg Val Val Gly Gly1 5 3538PRTArtificial Sequenceu-PA
cleavage site 353Pro Gly Ala Arg Gly Arg Ala Phe1 5
3548PRTArtificial Sequenceu-PA cleavage site 354Ser Ser Ser Arg Gly
Pro Thr His1 5 3558PRTArtificial Sequenceu-PA cleavage site 355Val
Ser Asn Lys Tyr Phe Ser Asn1 5 3568PRTArtificial Sequenceu-PA
cleavage site 356Asn Ser Gly Arg Ala Val Thr Tyr1 5
3578PRTArtificial Sequenceu-PA cleavage site 357Thr Tyr Ser Arg Ser
Arg Tyr Leu1 5 3588PRTArtificial Sequenceu-PA cleavage site 358Asn
Ser Gly Arg Ala Val Thr Tyr1 5 3598PRTArtificial Sequenceu-PA
cleavage site 359Pro Ser Gly Arg Gly Arg Thr Leu1 5
3608PRTArtificial Sequenceu-PA cleavage site 360Ala Gly Ser Arg Ala
Val Tyr Tyr1 5 3618PRTArtificial Sequenceu-PA cleavage site 361Thr
Tyr Gly Arg Ser Arg Thr Asn1 5 3628PRTArtificial Sequenceu-PA
cleavage site 362Asn Ser Ser Arg Gly Val Tyr Leu1 5
3638PRTArtificial Sequenceu-PA cleavage site 363Pro Ser Ser Arg Ser
Val Tyr Asn1 5 3648PRTArtificial Sequenceu-PA cleavage site 364Ala
Ser Gly Arg Gly Arg Thr Tyr1 5 3658PRTArtificial Sequenceu-PA
cleavage site 365Thr Ser Ser Arg Ala Val Tyr Leu1 5
3668PRTArtificial Sequenceu-PA cleavage site 366Asn Ser Gly Arg Ser
Arg Thr Leu1 5 3678PRTArtificial Sequenceu-PA cleavage site 367Val
Ser Gly Arg Ile Arg Thr Gly1 5 3688PRTArtificial Sequenceu-PA
cleavage site 368Ser Ser Gly Arg Ile Arg Thr Val1 5
3698PRTArtificial SequenceConsensus sequence for t-PA cleavage
siteVARIANT1, 2, 3, 5, 6, 7, 8Positions 1, 2, 3, 5, 6, 7, and 8 are
independantly any amino acidVARIANT4Position 4 is K or R 369Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 3708PRTArtificial Sequencet-PA cleavage
site 370Asn Ala Leu Arg Tyr Ala Pro Asp1 5 3718PRTArtificial
Sequencet-PA cleavage site 371Cys Pro Gly Arg Val Val Gly Gly1 5
3728PRTArtificial Sequencet-PA cleavage site 372Pro Gln Phe Arg Ile
Lys Gly Gly1 5 3738PRTArtificial Sequencet-PA cleavage site 373Ala
Leu Ser Arg Met Ala Val Leu1 5 3746PRTArtificial SequenceConsensus
sequence for Tryptase-epsilon cleavage siteVARIANT1Position 1 is K
or RVARIANT2, 3, 4, 5Positions 2, 3, 4, and 5 are independantly an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
MVARIANT6Position 6 is D or E 374Xaa Xaa Xaa Xaa Xaa Xaa1 5
3756PRTArtificial SequenceTryptase-epsilon cleavage site 375Arg Val
Val Gly Gly Glu1 5 3766PRTArtificial SequenceTryptase-epsilon
cleavage site 376Arg Ile Val Gly Gly Glu1 5 3776PRTArtificial
SequenceTryptase-epsilon cleavage site 377Arg Ile Ile Gly Gly Glu1
5 3786PRTArtificial SequenceTryptase-epsilon cleavage site 378Arg
Val Val Gly Gly Asp1 5 3796PRTArtificial SequenceTryptase-epsilon
cleavage site 379Arg Ile Val Gly Gly Asp1 5 3806PRTArtificial
SequenceTryptase-epsilon cleavage site 380Arg Ile Ile Gly Gly Asp1
5 3816PRTArtificial SequenceTryptase-epsilon cleavage site 381Lys
Val Val Gly Gly Glu1 5 3826PRTArtificial SequenceTryptase-epsilon
cleavage site 382Lys Ile Val Gly Gly Glu1 5 3836PRTArtificial
SequenceTryptase-epsilon cleavage site 383Lys Ile Ile Gly Gly Glu1
5 3846PRTArtificial SequenceTryptase-epsilon cleavage site 384Lys
Val Val Gly Gly Asp1 5 3856PRTArtificial SequenceTryptase-epsilon
cleavage site 385Lys Ile Val Gly Gly Asp1 5 3866PRTArtificial
SequenceTryptase-epsilon cleavage site 386Lys Ile Ile Gly Gly Asp1
5 3878PRTArtificial SequenceConsensus sequence for mMCP-7 cleavage
siteVARIANT1, 5, 6, 7, 8Positions 1, 5, 6, 7, and 8 are
independently any amino acidVARIANT2Potition 2 is an amidic amino
acid like N or Q, or an aliphatic hydrophobic amino acid like, G,
P, A, V, L, I, and MVARIANT3Position 3 is an aliphatic hydrophobic
amino acid like, G, P, A, V, L, I, and MVARIANT4Position 4 is K or
R 387Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3888PRTArtificial
SequencemMCP-7 cleavage site 388Leu Ser Ser Arg Gln Ser Pro Gly1 5
3898PRTArtificial SequencemMCP-7 cleavage site 389Leu Gln Ala Arg
Gly Ala Ser Leu1 5 3908PRTArtificial SequencemMCP-7 cleavage site
390Leu Gly Pro Lys Ala Ile Thr Met1 5 3918PRTArtificial
SequencemMCP-7 cleavage site 391Leu Gly Pro Arg Ser Ala Val Tyr1 5
3928PRTArtificial SequenceConsensus sequence for ECE-1 cleavage
siteVARIANT1, 2, 3, 4, 6, 7, 8Positions 1, 2, 3, 4, 6, 7, and 8 are
independantly any amino acidVARIANT5Position 5 is F, L, I, V, or Y
392Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3938PRTArtificial
SequenceECE-1 cleavage site 393His Gln Lys Leu Val Phe Phe Ala1 5
3948PRTArtificial SequenceECE-1 cleavage site 394His His Gln Lys
Leu Val Phe Phe1 5 3958PRTArtificial SequenceECE-1 cleavage site
395Lys Leu Val Phe Phe Ala Glu Asp1 5 3968PRTArtificial
SequenceECE-1 cleavage site 396Asp Arg Val Tyr Ile His Pro Phe1 5
3978PRTArtificial SequenceECE-1 cleavage site 397Tyr Ile His Pro
Phe His Leu Val1 5 3988PRTArtificial SequenceECE-1 cleavage site
398Tyr Gly Leu Gly Ser Pro Arg Ser1 5 3998PRTArtificial
SequenceECE-1 cleavage site 399Thr Pro Glu His Val Val Pro Tyr1 5
4008PRTArtificial SequenceECE-1 cleavage site 400Asp Ile Ile Trp
Val Asn Thr Pro1 5
4018PRTArtificial SequenceECE-1 cleavage site 401Asp Ile Ile Trp
Ile Asn Thr Pro1 5 4028PRTArtificial SequenceECE-1 cleavage site
402Cys His Leu Asp Ile Ile Trp Val1 5 4038PRTArtificial
SequenceECE-1 cleavage site 403His Leu Asp Ile Ile Trp Val Asn1 5
4048PRTArtificial SequenceECE-1 cleavage site 404Cys Val Tyr Phe
Cys His Leu Asp1 5 4058PRTArtificial SequenceECE-1 cleavage site
405Ser Cys Ser Ser Leu Met Asp Lys1 5 4068PRTArtificial
SequenceECE-1 cleavage site 406Glu Cys Val Tyr Phe Cys His Leu1 5
4078PRTArtificial SequenceECE-1 cleavage site 407Arg Ser Lys Arg
Cys Ser Cys Ser1 5 4088PRTArtificial SequenceECE-1 cleavage site
408Arg Ser Lys Arg Ala Leu Glu Asn1 5 4098PRTArtificial
SequenceECE-1 cleavage site 409Gly Phe Ser Pro Phe Arg Ser Ser1 5
4108PRTArtificial SequenceECE-1 cleavage site 410Pro Arg Arg Pro
Tyr Ile Leu Pro1 5 4118PRTArtificial SequenceECE-1 cleavage site
411Lys Pro Gln Gln Phe Phe Gly Leu1 5 4128PRTArtificial
SequenceECE-1 cleavage site 412Pro Gln Gln Phe Phe Gly Leu Met1 5
4138PRTArtificial SequenceConsensus sequence for KBGP cleavage
siteVARIANT1Position 1 is an acidic amino acid like D and
EVARIANT2, 5Positions 2 and 5 are independantly T or an aliphatic
hydrophobic amino acid like, G, P, A, V, L, I, and MVARIANT3,
8Positions 3 and 8 are independantly an aliphatic hydrophobic amino
acid like, G, P, A, V, L, I, and MVARIANT4Position 4 is an aromatic
amino acid like F, WVARIANT6Position 6 is an amidic amino acid like
N and QVARIANT(7)...(7)Position 7 is an uncharged amino acid like
C, S, and T, or a C-beta branched amino acid like I, V, or T 413Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 4148PRTArtificial SequenceKBGP
cleavage site 414Asp Ile Ile Trp Val Asn Thr Pro1 5
4158PRTArtificial SequenceKBGP cleavage site 415Asp Ile Ile Trp Ile
Asn Thr Pro1 5 4168PRTArtificial SequenceConsensus sequence for
Cathepsin L cleavage siteVARIANT1Position 1 is W, D, E, N, Q, H, K,
R, C, S, T, G, P, A, V, L, I or MVARIANT2, 4, 5, 6, 7, 8Positions
2, 4, 5, 6, 7, 8 are independantly any amino acidVARIANT3Position 3
is L, V, F or Y 416Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
4178PRTArtificial SequenceCathepsin L cleavage site 417Met Phe Leu
Glu Ala Ile Pro Met1 5 4188PRTArtificial SequenceCathepsin L
cleavage site 418Lys Val Phe Gln Glu Pro Leu Phe1 5
4198PRTArtificial SequenceCathepsin L cleavage site 419Ala Thr Leu
Thr Phe Asp His Ser1 5 4208PRTArtificial SequenceCathepsin L
cleavage site 420Pro Leu Phe Tyr Glu Ala Pro Arg1 5
4218PRTArtificial SequenceCathepsin L cleavage site 421Thr Gly Leu
Arg Asp Pro Phe Asn1 5 4228PRTArtificial SequenceCathepsin L
cleavage site 422Lys Ile Leu His Leu Pro Thr Ser1 5
4238PRTArtificial SequenceCathepsin L cleavage site 423Ala His Leu
Lys Asn Ser Gln Glu1 5 4248PRTArtificial SequenceCathepsin L
cleavage site 424Ala Pro Leu Thr Ala Glu Ile Gln1 5
4258PRTArtificial SequenceCathepsin L cleavage site 425Glu Ala Leu
Phe Ala Glu Arg Lys1 5 4268PRTArtificial SequenceCathepsin L
cleavage site 426Glu Pro Leu Ala Ala Glu Arg Lys1 5
4278PRTArtificial SequenceCathepsin L cleavage site 427Gly Thr Phe
Thr Ser Asp Tyr Ser1 5 4288PRTArtificial SequenceCathepsin L
cleavage site 428Lys Tyr Leu Asp Ser Arg Arg Ala1 5
4298PRTArtificial SequenceCathepsin L cleavage site 429Gln Asp Phe
Val Gln Trp Leu Met1 5 4308PRTArtificial SequenceCathepsin L
cleavage site 430Lys Gln Leu Ala Thr Lys Ala Ala1 5
4318PRTArtificial SequenceCathepsin L cleavage site 431Ser Thr Phe
Glu Glu Arg Ser Tyr1 5 4328PRTArtificial SequenceCathepsin L
cleavage site 432Leu Arg Leu Glu Trp Pro Tyr Gln1 5
4338PRTArtificial SequenceCathepsin L cleavage site 433Arg Gly Phe
Phe Tyr Thr Pro Lys1 5 4348PRTArtificial SequenceCathepsin L
cleavage site 434Gly Phe Phe Tyr Thr Pro Lys Ala1 5
4358PRTArtificial SequenceCathepsin L cleavage site 435His Phe Phe
Lys Asn Ile Val Thr1 5 4368PRTArtificial SequenceCathepsin L
cleavage site 436Arg Gly Leu Ser Leu Ser Arg Phe1 5
4378PRTArtificial SequenceCathepsin L cleavage site 437Gln Trp Leu
Gly Ala Pro Val Pro1 5 4388PRTArtificial SequenceCathepsin L
cleavage site 438Asn Met Leu Lys Arg Gly Leu Pro1 5
4398PRTArtificial SequenceCathepsin L cleavage site 439Leu Ser Leu
Ala His Thr His Gln1 5 4408PRTArtificial SequenceCathepsin L
cleavage site 440Thr Pro Phe Ala Ala Thr Ser Ser1 5
4418PRTArtificial SequenceCathepsin L cleavage site 441Lys Leu Leu
Ala Val Ser Gly Pro1 5 4428PRTArtificial SequenceCathepsin L
cleavage site 442Gln Leu Phe Arg Arg Ala Val Leu1 5
4438PRTArtificial SequenceCathepsin L cleavage site 443Pro Arg Phe
Lys Ile Ile Gly Gly1 5 4446PRTArtificial SequenceConsensus sequence
for PAR1 cleavage siteVARIANT1Position 1 is a small non-polar amino
acid like A, C G, S, and TVARIANT2, 3Positions 2 and 3 are
independantly a large non-polar amino acid like F, I, L, M, V, or
an aromatic amino acid like F, H, W, or YVARIANT4Position 4 is an
aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
MVARIANT5Position 5 is K or RVARIANT6Position 6 is an amidic amino
acid like N and Q, or an aromatic hydrophobic amino acid like F, W,
or Y 444Xaa Xaa Xaa Xaa Xaa Xaa1 5 4456PRTArtificial SequencePAR1
cleavage site 445Ser Phe Leu Leu Arg Asn1 5 4466PRTArtificial
SequencePAR1 cleavage site 446Ser Phe Phe Leu Arg Asn1 5
4476PRTArtificial SequencePAR1 cleavage site 447Ser Phe Phe Leu Lys
Asn1 5 4486PRTArtificial SequencePAR1 cleavage site 448Thr Phe Leu
Leu Arg Asn1 5 4496PRTArtificial SequencePAR1 cleavage site 449Gly
Phe Pro Gly Lys Phe1 5 4506PRTArtificial SequencePAR1 cleavage site
450Gly Tyr Pro Ala Lys Phe1 5 4516PRTArtificial SequencePAR1
cleavage site 451Gly Tyr Pro Leu Lys Phe1 5 4526PRTArtificial
SequencePAR1 cleavage site 452Gly Tyr Pro Ile Lys Phe1 5
4536PRTArtificial SequenceConsensus sequence for PAR2 cleavage
siteVARIANT1Position 1 is a small non-polar amino acid like A, C G,
S, and TVARIANT2, 3, 6Positions 2, 3, and 6 are independantly a
large non-polar amino acid like F, I, L, M, VVARIANT4Position 4 is
an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and
MVARIANT5Position 5 is K or R 453Xaa Xaa Xaa Xaa Xaa Xaa1 5
4546PRTArtificial SequencePAR2 cleavage site 454Ser Leu Ile Gly Lys
Val1 5 4556PRTArtificial SequencePAR2 cleavage site 455Ser Leu Ile
Gly Arg Leu1 5 4566PRTArtificial SequenceConsensus sequence for
PAR3 cleavage siteVARIANT1, 4Positions 1 and 4 are independantly a
small non-polar amino acid like A, C G, S, and TVARIANT2Position 2
is a large non-polar amino acid like F, I, L, M, VVARIANT3Position
3 is an amidic amino acid like N and Q, or a basic amino acid like
K and RVARIANT5Position 5 is a small non-polar amino acid like A, C
G, S, and T, or a small polar amino acid like D, N, or
PVARIANT6Position 6 is an acidic amino acid like D and E, or a
small polar amino acid like D, N, or P 456Xaa Xaa Xaa Xaa Xaa Xaa1
5 4576PRTArtificial SequencePAR3 cleavage site 457Thr Phe Arg Gly
Ala Pro1 5 4586PRTArtificial SequencePAR3 cleavage site 458Ser Phe
Asn Gly Gly Pro1 5 4596PRTArtificial SequencePAR3 cleavage site
459Ser Phe Asn Gly Asn Glu1 5 4606PRTArtificial SequenceConsensus
sequence for PAR4 cleavage siteVARIANT1Position 1 is a small
non-polar amino acid like A, C G, S, and TVARIANT2Position 2 is a
large non-polar amino acid like F, I, L, M, V, or an aromatic amino
acid like F, H, W, or YVARIANT3, 4Positions 3 and 4 are
independantly an aliphatic hydrophobic amino acid like, G, P, A, V,
L, I, and MVARIANT5Position 5 is K or RVARIANT6Position 6 is a
basic amino acid like K and R, an aromatic hydrophobic amino acid
like F, W, or Y, or an aliphatic hydrophobic amino acid like, G, P,
A, V, L, I, and M 460Xaa Xaa Xaa Xaa Xaa Xaa1 5 4616PRTArtificial
SequencePAR4 cleavage site 461Gly Tyr Pro Gly Gln Val1 5
4626PRTArtificial SequencePAR4 cleavage site 462Ala Tyr Pro Gly Lys
Phe1 5 4636PRTArtificial SequencePAR4 cleavage site 463Thr Tyr Pro
Gly Lys Phe1 5 4646PRTArtificial SequencePAR4 cleavage site 464Gly
Tyr Pro Gly Lys Tyr1 5 4656PRTArtificial SequencePAR4 cleavage site
465Gly Tyr Pro Gly Lys Trp1 5 4666PRTArtificial SequencePAR4
cleavage site 466Gly Tyr Pro Gly Lys Lys1 5 4676PRTArtificial
SequencePAR4 cleavage site 467Gly Tyr Pro Gly Lys Phe1 5
4686PRTArtificial SequencePAR4 cleavage site 468Gly Tyr Pro Gly Arg
Phe1 5 4696PRTArtificial SequencePAR4 cleavage site 469Gly Tyr Pro
Gly Phe Lys1 5 4706PRTArtificial SequencePAR4 cleavage site 470Gly
Tyr Pro Ala Lys Phe1 5 4716PRTArtificial SequencePAR4 cleavage site
471Gly Phe Pro Gly Lys Phe1 5 4726PRTArtificial SequencePAR4
cleavage site 472Gly Phe Pro Gly Lys Pro1 5 4736PRTArtificial
SequencePAR4 cleavage site 473Ser Tyr Pro Gly Lys Phe1 5
4746PRTArtificial SequencePAR4 cleavage site 474Ser Tyr Pro Ala Lys
Phe1 5 4756PRTArtificial SequencePAR4 cleavage site 475Ser Tyr Pro
Gly Arg Phe1 5 4766PRTArtificial SequencePAR4 cleavage site 476Ser
Tyr Ala Gly Lys Phe1 5 4776PRTArtificial SequencePAR4 cleavage site
477Ser Phe Pro Gly Gln Pro1 5 4786PRTArtificial SequencePAR4
cleavage site 478Ser Phe Pro Gly Gln Ala1 5 4798PRTArtificial
SequenceADAMTS13 cleavage site 479Asn Leu Val Tyr Met Val Thr Gly1
5 4805PRTArtificial SequenceBovine enterokinase protease cleavage
site 480Asp Asp Asp Asp Lys1 5 4817PRTArtificial SequenceConsensus
sequence for TEV protease cleavage siteVARIANT2, 3, 5Positions 2,
3, and 5 are independantly any amino amino acid 481Glu Xaa Xaa Tyr
Xaa Gln Gly1 5 4827PRTArtificial SequenceConsensus sequence for TEV
protease cleavage siteVARIANT2, 3, 5Positions 2, 3, and 5 are
independantly any amino acid 482Glu Xaa Xaa Tyr Xaa Gln Ser1 5
4837PRTArtificial SequenceTEV protease cleavage site 483Glu Asn Leu
Tyr Phe Gln Gly1 5 4847PRTArtificial SequenceTEV protease cleavage
site 484Glu Asn Leu Tyr Phe Gln Ser1 5 4857PRTArtificial
SequenceTEV protease cleavage site 485Glu Asn Ile Tyr Thr Gln Gly1
5 4867PRTArtificial SequenceTEV protease cleavage site 486Glu Asn
Ile Tyr Thr Gln Ser1 5 4877PRTArtificial SequenceTEV protease
cleavage site 487Glu Asn Ile Tyr Leu Gln Gly1 5 4887PRTArtificial
SequenceTEV protease cleavage site 488Glu Asn Ile Tyr Leu Gln Ser1
5 4897PRTArtificial SequenceTEV protease cleavage site 489Glu Asn
Val Tyr Phe Gln Gly1 5 4907PRTArtificial SequenceTEV protease
cleavage site 490Glu Asn Val Tyr Ser Gln Ser1 5 4917PRTArtificial
SequenceTEV protease cleavage site 491Glu Asn Val Tyr Ser Gln Gly1
5 4927PRTArtificial SequenceTEV protease cleavage site 492Glu Asn
Val Tyr Ser Gln Ser1 5 4937PRTArtificial SequenceConsensus sequence
for TVMV protease cleavage siteVARIANT1, 2Positions 1 and 2 are
independantly any amino acid 493Xaa Xaa Val Arg Phe Gln Gly1 5
4947PRTArtificial SequenceConsensus sequence for TVMV protease
cleavage siteVARIANT1, 2Positions 1 and 2 are independantly any
amino acid 494Xaa Xaa Val Arg Phe Gln Ser1 5 4957PRTArtificial
SequenceTVMV protease cleavage site 495Glu Thr Val Arg Phe Gln Gly1
5 4967PRTArtificial SequenceTVMV protease cleavage site 496Glu Thr
Val Arg Phe Gln Ser1 5 4977PRTArtificial SequenceTVMV protease
cleavage site 497Asn Asn Val Arg Phe Gln Gly1 5 4987PRTArtificial
SequenceTVMV protease cleavage site 498Asn Asn Val Arg Phe Gln Ser1
5 4997PRTArtificial SequenceConsensus sequence for HR3C protease
cleavage siteVARIANT1Position 1 can be amino acid, with D or E
preferredVARIANT2Position 2 is S, T, and an aliphatic hydrophobic
amino acid like G, P, A, V, L, I, and M 499Xaa Xaa Leu Phe Gln Gly
Pro1 5 5007PRTArtificial SequenceHR3C protease cleavage site 500Glu
Ala Leu Phe Gln Gly Pro1 5 5017PRTArtificial SequenceHR3C protease
cleavage site 501Glu Val Leu Phe Gln Gly Pro1 5 5027PRTArtificial
SequenceHR3C protease cleavage site 502Glu Leu Leu Phe Gln Gly Pro1
5 5037PRTArtificial SequenceHR3C protease cleavage site 503Asp Ala
Leu Phe Gln Gly Pro1 5 5047PRTArtificial SequenceHR3C protease
cleavage site 504Asp Val Leu Phe Gln Gly Pro1 5 5057PRTArtificial
SequenceHR3C protease cleavage site 505Asp Leu Leu Phe Gln Gly Pro1
5 5066PRTArtificial SequenceConsensus sequence for Subtilisin
protease cleavage siteVARIANT1, 2, 3, 4Positions 1, 2, 3, and 4 are
independantly any amino acid 506Xaa Xaa Xaa Xaa His Tyr1 5
5076PRTArtificial SequenceConsensus sequence for Subtilisin
protease cleavage siteVARIANT1, 2, 3, 4Positions 1, 2, 3, and 4 are
independantly any amino acid 507Xaa Xaa Xaa Xaa Tyr His1 5
5082PRTArtificial SequenceSubtilisin protease cleavage site 508His
Tyr1 5092PRTArtificial SequenceSubtilisin protease cleavage site
509Tyr His1 5106PRTArtificial SequenceSubtilisin protease cleavage
site 510Pro Gly Ala Ala His Tyr1 5 5116PRTArtificial
SequenceHydroxylamine cleavage site 511Asn Gly Asn Gly Asn Gly1 5
5122PRTArtificial SequenceHydroxylamine cleavage site 512Asn Gly1
5135PRTArtificial SequenceConsensus sequence for SUMO/ULP-1
protease cleavage siteVARIANT3, 4, 5Positions 3, 4, and 5 are
independantly any amino acid 513Gly Gly Xaa Xaa Xaa1 5
51498PRTArtificial SequenceSUMO/ULP-1 protease cleavage site 514Met
Ala Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro1 5 10
15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser
20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg
Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met
Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala
Asp Gln Thr Pro Glu Asp65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile
Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly5155PRTArtificial
SequenceConsensus sequence for Caspase 3 protease cleavage
siteVARIANT2Position 2 can be any amino acid with E
preferredVARIANT3Position 3 is any amino acidVARIANT5Position 5 can
be any amino acid with G or S preferred 515Asp Xaa Xaa Asp Xaa1 5
5165PRTArtificial SequenceCaspase 3 protease cleavage site 516Asp
Glu Val Asp Gly1 5 5175PRTArtificial SequenceCaspase 3 protease
cleavage site 517Asp Glu Val Asp Ser1 5 5185PRTArtificial
SequenceCaspase 3 protease cleavage site 518Asp Glu Pro Asp Gly1 5
5195PRTArtificial SequenceCaspase 3 protease cleavage site 519Asp
Glu Pro Asp Ser1 5 5205PRTArtificial SequenceCaspase 3 protease
cleavage site 520Asp Glu Leu Asp Gly1 5 5215PRTArtificial
SequenceCaspase 3 protease cleavage site 521Asp Glu Leu Asp Ser1 5
5224PRTArtificial SequenceFlexible G-spacer 522Gly Gly Gly Gly1
5235PRTArtificial SequenceFlexible G-spacer 523Gly Gly Gly Gly Ser1
5 5244PRTArtificial SequenceFlexible A-spacer 524Ala Ala Ala Ala1
5255PRTArtificial SequenceFlexible A-spacer 525Ala Ala Ala Ala Val1
5 5263939DNAArtificial SequenceDNA encoding a BoNT/A with a HIS tag
526atgccgttcg taaacaaaca gttcaactat aaagacccag
tcaacggcgt ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga
aagcatttaa aatccataac 120aaaatttggg tgatcccgga gcgcgatacg
ttcacgaacc cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca
ggtcccggtg tcttactatg atagcacata cctgagtacc 240gacaatgaaa
aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg
300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt
ttggggtggt 360agcaccatcg atacagaact caaagtgatt gacaccaact
gcatcaatgt gattcagcct 420gatgggagct accggtccga agagcttaac
ctcgtaatca ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc
ttttgggcat gaagtcctga atctgacgcg gaatggctat 540ggatcgacgc
agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc
600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc
agcggtgacc 660ttggcacatg aacttattca tgccgggcat cgcttgtatg
gaatcgccat taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat
tacgagatgt cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg
cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840gaatttcggc
tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg
900aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt
caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg
ataaactgaa atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca
gaggataact ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct
gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140aaagttaact
acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac
1200tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa
aaattttacc 1260ggcttgtttg aattttataa gctcctgtgt gtccgcggta
ttatcaccag caaaaccaaa 1320tccttggata agggctataa caaggcgctc
aatgatttat gcatcaaggt gaacaactgg 1380gacttgtttt tctctccatc
tgaagataat tttactaacg acttgaacaa aggagaggaa 1440attacttccg
ataccaacat cgaagcagcg gaagagaata ttagcctgga tcttattcaa
1500caatattacc tgacctttaa ttttgataac gagcctgaga acatttccat
tgagaatctc 1560agctctgaca tcatcggcca gctggaactg atgccgaata
tcgaacgctt tcctaatgga 1620aagaaatatg aattggacaa atacaccatg
ttccactatc tccgcgcgca ggagtttgag 1680cacggcaagt ctcgtattgc
tctgaccaat tcggtaaacg aagccctttt aaatccttcg 1740cgtgtgtaca
cctttttctc aagcgattat gttaaaaaag tgaacaaggc gaccgaagcg
1800gcgatgtttt tgggatgggt ggaacaactg gtatatgact ttacggatga
aacttctgaa 1860gtctcgacca ccgacaaaat tgccgatatt accattatca
ttccctatat tggccctgca 1920ctgaacattg gtaacatgct gtataaagat
gattttgtgg gcgccctgat cttttcaggc 1980gctgttatcc tgctggaatt
tatcccggaa atcgccattc cagtactcgg tacctttgcg 2040ctggtgtcct
atatcgcaaa caaagttttg actgtccaga cgatcgacaa cgcgctcagt
2100aaacgtaacg aaaaatggga tgaggtgtat aagtatattg ttaccaactg
gctcgctaaa 2160gtaaacaccc agattgacct gattcgcaag aagatgaaag
aagcgctgga aaaccaagca 2220gaagcgacca aagctattat caactatcaa
tataaccagt acacagagga agaaaagaat 2280aacatcaact tcaacatcga
cgacttatct tcaaagctga atgaatctat taacaaagcg 2340atgattaata
ttaacaagtt cttgaaccaa tgtagtgtca gctatctgat gaactcgatg
2400atcccttacg gtgtgaaacg tctggaagac ttcgatgcaa gccttaaaga
tgcccttctg 2460aagtatattt acgataatcg cggaactctt attggccaag
tggatcgctt aaaagataaa 2520gtcaacaaca cgctgagtac agacatccct
tttcagctgt ctaaatatgt ggacaatcag 2580cgcctgctgt ccacgtttac
ggaatacatc aaaaacatca tcaacactag tattctgaac 2640ttgcgttacg
agagtaacca tctgattgat ctgagccgtt acgcatctaa aatcaacatc
2700ggctcgaagg tgaacttcga tcctatcgac aaaaaccaga ttcaattgtt
caacttagaa 2760tcgtcaaaga ttgaagttat cttaaaaaat gcgattgtat
ataattcaat gtacgaaaat 2820ttctctacga gcttttggat tcgtattccg
aaatatttca acagtatctc tttaaacaac 2880gagtatacta tcatcaattg
tatggagaat aacagcgggt ggaaagtgag ccttaactat 2940ggtgaaatca
tctggactct gcaggacact caagaaatta aacaacgcgt ggtgtttaaa
3000tactcacaga tgattaacat ctcggattat attaatcgct ggatttttgt
gacaattact 3060aacaaccggc tgaacaacag caaaatttac attaacggtc
gcctgatcga tcagaaacca 3120atcagtaatc tcggtaacat tcacgcatcg
aataatatca tgttcaaact ggatggttgt 3180cgcgacacgc accgttacat
ttggatcaaa tacttcaatt tattcgacaa agaactcaac 3240gaaaaggaga
ttaaggatct ttatgacaat cagtctaatt cgggtattct gaaagacttt
3300tggggtgatt accttcagta cgataaaccg tattatatgt taaacttata
tgatccgaat 3360aaatacgttg acgtcaacaa cgttggcatt cgtggctata
tgtatctgaa agggccgcgt 3420ggcagcgtga tgaccactaa catttactta
aactcctccc tctatcgcgg tactaaattt 3480attatcaaga aatatgcctc
tggcaacaag gacaatatcg tacgcaataa cgatcgcgtc 3540tacattaacg
tggtggtgaa gaataaagaa tatcgtctgg cgaccaatgc tagtcaggcg
3600ggcgtggaga aaattctgtc tgcacttgaa atcccggatg tgggtaattt
atcccaggtg 3660gttgtgatga aaagtaaaaa tgaccaaggg atcaccaata
aatgcaaaat gaatctgcaa 3720gataacaacg gcaacgacat tggttttatc
ggcttccacc aattcaataa tatcgcgaaa 3780ctggtggcct caaattggta
caaccgtcag attgagcgca gctcccgcac tttaggctgt 3840agctgggagt
tcattccggt agatgacggt tggggagaac gcccattgaa agtcgacaag
3900cttgcggccg cactcgagca ccaccaccac caccactga
39395271312PRTArtificial SequenceBoNT/A with a HIS tag 527Met Pro
Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15
Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20
25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu
Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro
Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser
Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys
Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu
Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp
Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg
Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150
155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu
Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser
Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp
Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro
Ala Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala Gly His Arg
Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240 Arg Val Phe Lys
Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val
Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270
Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275
280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile
Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe
Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys
Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met
Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe
Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala
Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 Thr Ile
Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395
400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu
405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys
Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys
Gly Tyr Asn Lys 435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn
Asn Trp Asp Leu Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr
Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480 Ile Thr Ser Asp Thr
Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile
Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu
Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520
525 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
530 535 540 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu
Phe Glu545 550 555 560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser
Val Asn Glu Ala Leu 565 570 575 Leu Asn Pro Ser Arg Val Tyr Thr Phe
Phe Ser Ser Asp Tyr Val Lys 580 585 590 Lys Val Asn Lys Ala Thr Glu
Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605 Gln Leu Val Tyr Asp
Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620 Asp Lys Ile
Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640
Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645
650 655 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile
Ala 660 665 670 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile
Ala Asn Lys 675 680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu
Ser Lys Arg Asn Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile
Val Thr Asn Trp Leu Ala Lys705 710 715 720 Val Asn Thr Gln Ile Asp
Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala
Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr
Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765
Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770
775 780 Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser
Met785 790 795 800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp
Ala Ser Leu Lys 805 810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn
Arg Gly Thr Leu Ile Gly 820 825 830 Gln Val Asp Arg Leu Lys Asp Lys
Val Asn Asn Thr Leu Ser Thr Asp 835 840 845 Ile Pro Phe Gln Leu Ser
Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu
Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn865 870 875 880 Leu
Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890
895 Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn
900 905 910 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val
Ile Leu 915 920 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn
Phe Ser Thr Ser 930 935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn
Ser Ile Ser Leu Asn Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys
Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly
Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln
Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005
Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu
1010 1015 1020 Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp
Gln Lys Pro1025 1030 1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala
Ser Asn Asn Ile Met Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp
Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe
Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085
Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr
1090 1095 1100 Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr
Asp Pro Asn1105 1110 1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly
Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly Ser
Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr
Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165
Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val
1170 1175 1180 Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala
Ser Gln Ala1185 1190 1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu
Glu Ile Pro Asp Val Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val
Met Lys Ser Lys Asn Asp Gln Gly Ile Thr 1220 1225 1230 Asn Lys Cys
Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245
Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser
1250 1255 1260 Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr
Leu Gly Cys1265 1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp
Gly Trp Gly Glu Arg Pro Leu 1285 1290 1295 Lys Val Asp Lys Leu Ala
Ala Ala Leu Glu His His His His His His 1300 1305 1310
5283921DNAArtificial SequenceDNA encoding a BoNT/A-TEV with a HIS
tag 528atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt
ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa
aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc
cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg
tcttactatg atagcacata cctgagtacc 240gacaatgaaa aggacaacta
cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300acagatctcg
gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt
360agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt
gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca
ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat
gaagtcctga atctgacgcg gaatggctat 540ggatcgacgc agtatattcg
tttttctcca gatttcacat ttggatttga agaaagcctc 600gaagttgata
cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc
660ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat
taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt
cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat
gcaaaattta ttgatagtct gcaagaaaac 840gaatttcggc tgtactatta
caataaattc aaagacattg catcaacctt aaacaaggcg 900aaaagcattg
tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa
960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa
atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact
ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat
aaagccgtct ttaagatcaa catcgtaccg 1140aaagttaact acaccatcta
tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200tttaacggcc
agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc
1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag
caaaggcggt 1320ggttctggcg gtggtgaaaa cctgtacttc cagggcggtg
gctccggtgg tggtgcgctc 1380aatgatttat gcatcaaggt gaacaactgg
gacttgtttt tctctccatc tgaagataat 1440tttactaacg acttgaacaa
aggagaggaa attacttccg ataccaacat cgaagcagcg 1500gaagagaata
ttagtctaga tcttattcaa caatattacc tgacctttaa ttttgataac
1560gagcctgaga acatttccat tgagaatctc agctctgaca tcatcggcca
gctggaactg 1620atgccgaata tcgaacgctt tcctaatgga aagaaatatg
aattggacaa atacaccatg 1680ttccactatc tccgcgcgca ggagtttgag
cacggcaagt ctcgtattgc tctgaccaat 1740tcggtaaacg aagccctttt
aaatccttcg cgtgtgtaca cctttttctc aagcgattat 1800gttaaaaaag
tgaacaaggc gaccgaagcg gcgatgtttt tgggatgggt ggaacaactg
1860gtatatgact ttacggatga aacttctgaa gtctcgacca ccgacaaaat
tgccgatatt 1920accattatca ttccctatat tggccctgca ctgaacattg
gtaacatgct gtataaagat 1980gattttgtgg gcgccctgat cttttcaggc
gctgttatcc tgctggaatt tatcccggaa 2040atcgccattc cagtactcgg
tacctttgcg ctggtgtcct atatcgcaaa caaagttttg 2100actgtccaga
cgatcgacaa cgcgctcagt aaacgtaacg aaaaatggga tgaggtgtat
2160aagtatattg ttaccaactg gctcgctaaa gtaaacaccc agattgacct
gattcgcaag 2220aagatgaaag aagcgctgga aaaccaagca gaagcgacca
aagctattat caactatcaa 2280tataaccagt acacagagga agaaaagaat
aacatcaact tcaacatcga cgacttatct 2340tcaaagctga atgaatctat
taacaaagcg atgattaata ttaacaagtt cttgaaccaa 2400tgtagtgtca
gctatctgat gaactcgatg atcccttacg gtgtgaaacg tctggaagac
2460ttcgatgcaa gccttaaaga tgcccttctg aagtatattt acgataatcg
cggaactctt 2520attggccaag tggatcgctt aaaagataaa gtcaacaaca
cgctgagtac agacatccct 2580tttcagctgt ctaaatatgt ggacaatcag
cgcctgctgt ccacgtttac ggaatacatc 2640aaaaacatca tcaacactag
tattctgaac ttgcgttacg agagtaacca tctgattgat 2700ctgagccgtt
acgcatctaa aatcaacatc ggatccaagg tgaacttcga tcctatcgac
2760aaaaaccaga ttcaattgtt caacttagaa tcgtcaaaga ttgaagttat
cttaaaaaat 2820gcgattgtat ataattcaat gtacgaaaat ttctctacga
gcttttggat tcgtattccg 2880aaatatttca acagtatctc tttaaacaac
gagtatacta tcatcaattg tatggagaat 2940aacagcgggt ggaaagtgag
ccttaactat ggtgaaatca tctggactct gcaggacact 3000caagaaatta
aacaacgcgt ggtgtttaaa tactcacaga tgattaacat ctcggattat
3060attaatcgct ggatttttgt gacaattact aacaaccggc tgaacaacag
caaaatttac 3120attaacggtc gcctgatcga tcagaaacca atcagtaatc
tcggtaacat tcacgcatcg 3180aataatatca tgttcaaact ggatggttgt
cgcgacacgc accgttacat ttggatcaaa 3240tacttcaatt tattcgacaa
agaactcaac gaaaaggaga ttaaggatct ttatgacaat 3300cagtctaatt
cgggtattct gaaagacttt tggggtgatt accttcagta cgataaaccg
3360tattatatgt taaacttata tgatccgaat aaatacgttg acgtcaacaa
cgttggcatt 3420cgtggctata tgtatctgaa agggccgcgt ggcagcgtga
tgaccactaa catttactta 3480aactcctccc tctatcgcgg tactaaattt
attatcaaga aatatgcctc tggcaacaag 3540gacaatatcg tacgcaataa
cgatcgcgtc tacattaacg tggtggtgaa gaataaagaa 3600tatcgtctgg
cgaccaatgc tagtcaggcg ggcgtggaga aaattctgtc tgcacttgaa
3660atcccggatg tgggtaattt atcccaggtg gttgtgatga aaagtaaaaa
tgaccaaggg 3720atcaccaata aatgcaaaat gaatctgcaa gataacaacg
gcaacgacat tggttttatc 3780ggcttccacc aattcaataa tatcgcgaaa
ctggtggcct caaattggta caaccgtcag 3840attgagcgca gctcccgcac
tttaggctgt agctgggagt tcattccggt agatgacggt 3900tggggagaac
gcccattgta a 39215291306PRTArtificial SequenceBoNT/A-TEV with a HIS
tag 529Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn
Gly1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln
Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp
Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly
Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser
Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu Lys Asp
Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr
Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg
Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120
125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr
130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala
Asp Ile145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu
Val Leu Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr
Ile Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser
Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe
Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245
250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala
Lys 260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr
Tyr Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys
Ala Lys Ser Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met
Lys Asn Val Phe Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser Glu Asp
Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys
Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe
Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370
375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala
Asn385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn
Phe Thr Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr
Lys Leu Leu Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys Gly Gly
Gly Ser Gly Gly Gly Glu Asn Leu 435 440 445 Tyr Phe Gln Gly Gly Gly
Ser Gly Gly Gly Ala Leu Asn Asp Leu Cys 450 455 460 Ile Lys Val Asn
Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn465 470 475 480 Phe
Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn 485 490
495 Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln Gln Tyr
500 505 510 Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser
Ile Glu 515 520 525 Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu
Met Pro Asn Ile 530 535 540 Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
Leu Asp Lys Tyr Thr Met545 550 555 560 Phe His Tyr Leu Arg Ala Gln
Glu Phe Glu His Gly Lys Ser Arg Ile 565 570 575 Ala Leu Thr Asn Ser
Val Asn Glu Ala Leu Leu Asn Pro Ser Arg Val 580 585 590 Tyr Thr Phe
Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr 595 600 605 Glu
Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr Asp Phe 610 615
620 Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala Asp
Ile625 630 635 640 Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn
Ile Gly Asn Met 645 650 655 Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
Ile Phe Ser Gly Ala Val 660 665 670 Ile Leu Leu Glu Phe Ile Pro Glu
Ile Ala Ile Pro Val Leu Gly Thr 675 680 685 Phe Ala Leu Val Ser Tyr
Ile Ala Asn Lys Val Leu Thr Val Gln Thr 690 695 700 Ile Asp Asn Ala
Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu Val Tyr705 710 715 720 Lys
Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp 725 730
735 Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala
740 745 750 Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu
Glu Glu 755 760 765 Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser
Ser Lys Leu Asn 770 775 780 Glu Ser Ile Asn Lys Ala Met Ile Asn Ile
Asn Lys Phe Leu Asn Gln785 790 795 800 Cys Ser Val Ser Tyr Leu Met
Asn Ser Met Ile Pro Tyr Gly Val Lys 805 810 815 Arg Leu Glu Asp Phe
Asp Ala Ser Leu Lys Asp Ala Leu Leu Lys Tyr 820 825 830 Ile Tyr Asp
Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg Leu Lys 835 840 845 Asp
Lys Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser 850 855
860 Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Phe Thr Glu Tyr
Ile865 870 875 880 Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg
Tyr Glu Ser Asn 885 890 895 His Leu Ile Asp Leu Ser Arg Tyr Ala Ser
Lys Ile Asn Ile Gly Ser 900 905 910 Lys Val Asn Phe Asp Pro Ile Asp
Lys Asn Gln Ile Gln Leu Phe Asn 915 920 925 Leu Glu Ser Ser Lys Ile
Glu Val Ile Leu Lys Asn Ala Ile Val Tyr 930 935 940 Asn Ser Met Tyr
Glu Asn Phe Ser Thr Ser Phe Trp Ile Arg Ile Pro945 950 955 960 Lys
Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn 965 970
975 Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu
980 985 990 Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys Gln Arg
Val Val 995 1000 1005 Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp
Tyr Ile Asn Arg Trp 1010 1015 1020 Ile Phe Val Thr Ile Thr Asn Asn
Arg Leu Asn Asn Ser Lys Ile Tyr1025 1030 1035 1040Ile Asn Gly Arg
Leu Ile Asp Gln Lys Pro Ile Ser Asn Leu Gly Asn 1045 1050 1055 Ile
His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly Cys Arg Asp 1060
1065 1070 Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp
Lys Glu 1075 1080 1085 Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp
Asn Gln Ser Asn Ser 1090 1095 1100 Gly Ile Leu Lys Asp Phe Trp Gly
Asp Tyr Leu Gln Tyr Asp Lys Pro1105 1110 1115 1120Tyr Tyr Met Leu
Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn 1125 1130 1135 Asn
Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser 1140
1145 1150 Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg
Gly Thr 1155 1160 1165 Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn
Lys Asp Asn Ile Val 1170 1175 1180 Arg Asn Asn Asp Arg Val Tyr Ile
Asn Val Val Val Lys Asn Lys Glu1185 1190 1195 1200Tyr Arg Leu Ala
Thr Asn Ala Ser Gln Ala Gly Val Glu Lys Ile Leu 1205 1210 1215 Ser
Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln Val Val Val 1220
1225 1230 Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys Cys Lys
Met Asn 1235 1240 1245 Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe
Ile Gly Phe His Gln 1250 1255 1260 Phe Asn Asn Ile Ala Lys Leu Val
Ala Ser Asn Trp Tyr Asn Arg Gln1265 1270 1275 1280Ile Glu Arg Ser
Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro 1285 1290 1295 Val
Asp Asp Gly Trp Gly Glu Arg Pro Leu 1300 1305 5303945DNAArtificial
SequenceDNA encoding a BoNT/A comprising Thrombin (N880) and
Thrombin (D890) 530atgccgttcg taaacaaaca gttcaactat aaagacccag
tcaacggcgt ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga
aagcatttaa aatccataac 120aaaatttggg tgatcccgga gcgcgatacg
ttcacgaacc cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca
ggtcccggtg tcttactatg atagcacata cctgagtacc 240gacaatgaaa
aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg
300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt
ttggggtggt 360agcaccatcg atacagaact caaagtgatt gacaccaact
gcatcaatgt gattcagcct 420gatgggagct accggtccga agagcttaac
ctcgtaatca ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc
ttttgggcat gaagtcctga atctgacgcg gaatggctat 540ggatcgacgc
agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc
600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc
agcggtgacc 660ttggcacatg aacttattca tgccgggcat cgcttgtatg
gaatcgccat taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat
tacgagatgt cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg
cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840gaatttcggc
tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg
900aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt
caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg
ataaactgaa atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca
gaggataact ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct
gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140aaagttaact
acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac
1200tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa
aaattttacc 1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta
ttatcaccag caaaggcggt 1320ggttctggcg gtggtgaaaa cctgtacttc
cagggcggtg gctccggtgg tggtgcgctc 1380aatgatttat gcatcaaggt
gaacaactgg gacttgtttt tctctccatc tgaagataat 1440tttactaacg
acttgaacaa aggagaggaa attacttccg ataccaacat cgaagcagcg
1500gaagagaata ttagtctaga tcttattcaa caatattacc tgacctttaa
ttttgataac 1560gagcctgaga acatttccat tgagaatctc agctctgaca
tcatcggcca gctggaactg 1620atgccgaata tcgaacgctt tcctaatgga
aagaaatatg aattggacaa atacaccatg 1680ttccactatc tccgcgcgca
ggagtttgag cacggcaagt ctcgtattgc tctgaccaat 1740tcggtaaacg
aagccctttt aaatccttcg cgtgtgtaca cctttttctc aagcgattat
1800gttaaaaaag tgaacaaggc gaccgaagcg gcgatgtttt tgggatgggt
ggaacaactg 1860gtatatgact ttacggatga aacttctgaa gtctcgacca
ccgacaaaat tgccgatatt 1920accattatca ttccctatat tggccctgca
ctgaacattg gtaacatgct gtataaagat 1980gattttgtgg gcgccctgat
cttttcaggc gctgttatcc tgctggaatt tatcccggaa 2040atcgccattc
cagtactcgg tacctttgcg ctggtgtcct atatcgcaaa caaagttttg
2100actgtccaga cgatcgacaa cgcgctcagt aaacgtaacg aaaaatggga
tgaggtgtat 2160aagtatattg ttaccaactg gctcgctaaa gtaaacaccc
agattgacct gattcgcaag 2220aagatgaaag aagcgctgga aaaccaagca
gaagcgacca aagctattat caactatcaa 2280tataaccagt acacagagga
agaaaagaat aacatcaact tcaacatcga cgacttatct 2340tcaaagctga
atgaatctat taacaaagcg atgattaata ttaacaagtt cttgaaccaa
2400tgtagtgtca gctatctgat gaactcgatg atcccttacg gtgtgaaacg
tctggaagac 2460ttcgatgcaa gccttaaaga tgcccttctg aagtatattt
acgataatcg cggaactctt 2520attggccaag tggatcgctt aaaagataaa
gtcaacaaca cgctgagtac agacatccct 2580tttcagctgt ctaaatatgt
ggacaatcag cgcctgctgt ccacgtttac ggaatacatc 2640aaaaacatca
tcaacactag tattctgaac ctggtgccgc gtggctccta cgagagtaac
2700catctgattg atctggtgcc gcgtggcagc cgttacgcat ctaaaatcaa
catcggatcc 2760aaggtgaact tcgatcctat cgacaaaaac cagattcaat
tgttcaactt agaatcgtca 2820aagattgaag ttatcttaaa aaatgcgatt
gtatataatt caatgtacga aaatttctct 2880acgagctttt ggattcgtat
tccgaaatat ttcaacagta tctctttaaa caacgagtat 2940actatcatca
attgtatgga gaataacagc gggtggaaag tgagccttaa ctatggtgaa
3000atcatctgga ctctgcagga cactcaagaa attaaacaac gcgtggtgtt
taaatactca 3060cagatgatta acatctcgga ttatattaat cgctggattt
ttgtgacaat tactaacaac 3120cggctgaaca acagcaaaat ttacattaac
ggtcgcctga tcgatcagaa accaatcagt 3180aatctcggta acattcacgc
atcgaataat atcatgttca aactggatgg ttgtcgcgac 3240acgcaccgtt
acatttggat caaatacttc aatttattcg acaaagaact caacgaaaag
3300gagattaagg atctttatga caatcagtct aattcgggta ttctgaaaga
cttttggggt 3360gattaccttc agtacgataa accgtattat atgttaaact
tatatgatcc gaataaatac 3420gttgacgtca acaacgttgg cattcgcggc
tatatgtatc tgaaagggcc gcgtggcagc 3480gtgatgacca ctaacattta
cttaaactcc tccctctatc gcggtactaa atttattatc 3540aagaaatatg
cctctggcaa caaggacaat atcgtacgca ataacgatcg cgtctacatt
3600aacgtggtgg tgaagaataa agaatatcgt ctggcgacca atgctagtca
ggcgggcgtg 3660gagaaaattc tgtctgcact tgaaatcccg gatgtgggta
atttatccca ggtggttgtg 3720atgaaaagta aaaatgacca agggatcacc
aataaatgca aaatgaatct gcaagataac 3780aacggcaacg acattggttt
tatcggcttc caccaattca ataatatcgc gaagcttgtg 3840gcctcaaatt
ggtacaaccg tcagattgag cgcagctccc gcactttagg ctgtagctgg
3900gagttcattc cggtagatga cggttgggga gaacgcccat tgtaa
39455311314PRTArtificial SequenceBoNT/A comprising Thrombin (N880)
and Thrombin (D890) 531Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val Asn Gly1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro
Asn Ala Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His
Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn
Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln
Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80 Asp
Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90
95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val
100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu
Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro
Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145
150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn
Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe
Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val
Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp
Pro Ala Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala Gly His
Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240 Arg Val Phe
Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu
Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265
270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn
275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser
Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val
Phe Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly
Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys
Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe
Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys
Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 Thr
Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390
395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys
Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu
Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys Gly Gly Gly Ser Gly
Gly Gly Glu Asn Leu 435 440 445 Tyr Phe Gln Gly Gly Gly Ser Gly Gly
Gly Ala Leu Asn Asp Leu Cys 450 455 460 Ile Lys Val Asn Asn Trp Asp
Leu Phe Phe Ser Pro Ser Glu Asp Asn465 470 475 480 Phe Thr Asn Asp
Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn 485 490 495 Ile Glu
Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln Gln Tyr 500 505 510
Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu 515
520 525 Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met Pro Asn
Ile 530 535 540 Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys
Tyr Thr Met545 550 555 560 Phe His Tyr Leu Arg Ala Gln Glu Phe Glu
His Gly Lys Ser Arg Ile 565 570 575 Ala Leu Thr Asn Ser Val Asn Glu
Ala Leu Leu Asn Pro Ser Arg Val 580 585 590 Tyr Thr Phe Phe Ser Ser
Asp Tyr Val Lys Lys Val Asn Lys Ala Thr 595 600 605 Glu Ala Ala Met
Phe Leu Gly Trp Val Glu Gln Leu Val Tyr Asp Phe 610 615 620 Thr Asp
Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala Asp Ile625 630 635
640 Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met
645 650 655 Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe Ser Gly
Ala Val 660 665 670 Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro
Val Leu Gly Thr 675 680 685 Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys
Val Leu Thr Val Gln Thr 690 695 700 Ile Asp Asn Ala Leu Ser Lys Arg
Asn Glu Lys Trp Asp Glu Val Tyr705 710 715 720 Lys Tyr Ile Val Thr
Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp 725 730 735 Leu Ile Arg
Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala 740 745 750 Thr
Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu 755 760
765 Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys Leu Asn
770 775 780 Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu
Asn Gln785 790 795 800 Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile
Pro Tyr Gly Val Lys 805 810 815 Arg Leu Glu Asp Phe Asp Ala Ser Leu
Lys Asp Ala Leu Leu Lys Tyr 820 825 830 Ile Tyr Asp Asn Arg Gly Thr
Leu Ile Gly Gln Val Asp Arg Leu Lys 835 840 845 Asp Lys Val Asn Asn
Thr Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser 850 855 860 Lys Tyr Val
Asp Asn Gln Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile865 870 875 880
Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Val Pro Arg Gly Ser 885
890 895 Tyr Glu Ser Asn His Leu Ile Asp Leu Val Pro Arg Gly Ser Arg
Tyr 900 905 910 Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp
Pro Ile Asp 915 920 925 Lys Asn Gln Ile Gln Leu Phe Asn Leu Glu Ser
Ser Lys Ile Glu Val 930 935 940 Ile Leu Lys Asn Ala Ile Val Tyr Asn
Ser Met Tyr Glu Asn Phe Ser945 950 955 960 Thr Ser Phe Trp Ile Arg
Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu 965 970 975 Asn Asn Glu Tyr
Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp 980 985 990 Lys Val
Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr 995 1000
1005 Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile
Asn 1010 1015 1020 Ile Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr
Ile Thr Asn Asn1025 1030 1035 1040Arg Leu Asn Asn Ser Lys Ile Tyr
Ile Asn Gly Arg Leu Ile Asp Gln 1045 1050 1055 Lys Pro Ile Ser Asn
Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met 1060 1065 1070 Phe Lys
Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys 1075 1080
1085 Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys
Asp 1090 1095 1100 Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys
Asp Phe Trp Gly1105 1110 1115 1120Asp Tyr Leu Gln Tyr Asp Lys Pro
Tyr Tyr Met Leu Asn Leu Tyr Asp 1125 1130 1135 Pro Asn Lys Tyr Val
Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met 1140 1145 1150 Tyr Leu
Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu 1155 1160
1165 Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr
Ala 1170 1175 1180 Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp
Arg Val Tyr Ile1185 1190 1195 1200Asn Val Val Val Lys Asn Lys Glu
Tyr Arg Leu Ala Thr Asn Ala Ser 1205 1210 1215 Gln Ala Gly Val Glu
Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val 1220 1225 1230 Gly Asn
Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly 1235 1240
1245 Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn
Asp 1250 1255 1260 Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile
Ala Lys Leu Val1265 1270 1275 1280Ala Ser Asn Trp Tyr Asn Arg Gln
Ile Glu Arg Ser Ser Arg Thr Leu 1285 1290 1295 Gly Cys Ser Trp Glu
Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg 1300 1305 1310 Pro Leu
5323945DNAArtificial SequenceDNA encoding a BoNT/A comprising FXa
(N872) and Thrombin (E884) 532atgccgttcg taaacaaaca gttcaactat
aaagacccag tcaacggcgt ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg
cagcccgtga aagcatttaa aatccataac 120aaaatttggg tgatcccgga
gcgcgatacg ttcacgaacc cggaagaagg agatttaaac 180ccaccgcctg
aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc
240gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg
catttattcg 300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg
gcattccgtt ttggggtggt 360agcaccatcg atacagaact caaagtgatt
gacaccaact gcatcaatgt gattcagcct 420gatgggagct accggtccga
agagcttaac ctcgtaatca ttggcccgag cgcggatatt 480atccaattcg
aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat
540ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga
agaaagcctc 600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg
cgacggaccc agcggtgacc 660ttggcacatg aacttattca tgccgggcat
cgcttgtatg gaatcgccat taacccgaac 720cgtgttttca aggtgaatac
gaacgcgtat tacgagatgt cgggcttaga agtgtccttt 780gaagaactgc
gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac
840gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt
aaacaaggcg 900aaaagcattg tgggtaccac ggctagctta caatatatga
aaaacgtttt caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa
ttctctgtcg ataaactgaa atttgataaa 1020ctgtataaaa tgctcaccga
gatctacaca gaggataact ttgtcaaatt cttcaaggtc 1080ttgaatcgga
aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg
1140aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct
ggcggcgaac 1200tttaacggcc agaacaccga aatcaacaac atgaacttta
ctaaactgaa aaattttacc 1260ggcttgtttg aattctataa gctcctgtgt
gtccgcggta ttatcaccag caaaggcggt 1320ggttctggcg gtggtgaaaa
cctgtacttc cagggcggtg gctccggtgg tggtgcgctc 1380aatgatttat
gcatcaaggt gaacaactgg gacttgtttt tctctccatc tgaagataat
1440tttactaacg acttgaacaa aggagaggaa attacttccg ataccaacat
cgaagcagcg 1500gaagagaata ttagtctaga tcttattcaa caatattacc
tgacctttaa ttttgataac 1560gagcctgaga acatttccat tgagaatctc
agctctgaca tcatcggcca gctggaactg 1620atgccgaata tcgaacgctt
tcctaatgga aagaaatatg aattggacaa atacaccatg 1680ttccactatc
tccgcgcgca ggagtttgag cacggcaagt ctcgtattgc tctgaccaat
1740tcggtaaacg aagccctttt aaatccttcg cgtgtgtaca cctttttctc
aagcgattat 1800gttaaaaaag tgaacaaggc gaccgaagcg gcgatgtttt
tgggatgggt ggaacaactg 1860gtatatgact ttacggatga aacttctgaa
gtctcgacca ccgacaaaat tgccgatatt 1920accattatca ttccctatat
tggccctgca ctgaacattg gtaacatgct gtataaagat 1980gattttgtgg
gcgccctgat cttttcaggc gctgttatcc tgctggaatt tatcccggaa
2040atcgccattc cagtactcgg tacctttgcg ctggtgtcct atatcgcaaa
caaagttttg 2100actgtccaga cgatcgacaa cgcgctcagt aaacgtaacg
aaaaatggga tgaggtgtat 2160aagtatattg ttaccaactg gctcgctaaa
gtaaacaccc agattgacct gattcgcaag 2220aagatgaaag aagcgctgga
aaaccaagca gaagcgacca aagctattat caactatcaa 2280tataaccagt
acacagagga agaaaagaat aacatcaact tcaacatcga cgacttatct
2340tcaaagctga atgaatctat taacaaagcg atgattaata ttaacaagtt
cttgaaccaa 2400tgtagtgtca gctatctgat gaactcgatg atcccttacg
gtgtgaaacg tctggaagac 2460ttcgatgcaa gccttaaaga tgcccttctg
aagtatattt acgataatcg cggaactctt 2520attggccaag tggatcgctt
aaaagataaa gtcaacaaca cgctgagtac agacatccct 2580tttcagctgt
ctaaatatgt ggacaatcag cgcctgctgt ccacgtttac ggaatacatc
2640aaaaacattg agggccgtat caacactagt attctgaact tgcgttacga
gttggtgcca 2700cgcggttcta accatctgat tgatctgagc cgttacgcat
ctaaaatcaa catcggatcc 2760aaggtgaact tcgatcctat cgacaaaaac
cagattcaat tgttcaactt agaatcgtca 2820aagattgaag ttatcttaaa
aaatgcgatt gtatataatt caatgtacga aaatttctct 2880acgagctttt
ggattcgtat tccgaaatat ttcaacagta tctctttaaa caacgagtat
2940actatcatca attgtatgga gaataacagc gggtggaaag tgagccttaa
ctatggtgaa 3000atcatctgga ctctgcagga cactcaagaa attaaacaac
gcgtggtgtt taaatactca 3060cagatgatta acatctcgga ttatattaat
cgctggattt ttgtgacaat tactaacaac 3120cggctgaaca acagcaaaat
ttacattaac ggtcgcctga tcgatcagaa accaatcagt 3180aatctcggta
acattcacgc atcgaataat atcatgttca aactggatgg ttgtcgcgac
3240acgcaccgtt acatttggat caaatacttc aatttattcg acaaagaact
caacgaaaag 3300gagattaagg atctttatga caatcagtct aattcgggta
ttctgaaaga cttttggggt 3360gattaccttc agtacgataa accgtattat
atgttaaact tatatgatcc gaataaatac 3420gttgacgtca acaacgttgg
cattcgcggc tatatgtatc tgaaagggcc gcgtggcagc 3480gtgatgacca
ctaacattta cttaaactcc tccctctatc gcggtactaa atttattatc
3540aagaaatatg cctctggcaa caaggacaat atcgtacgca ataacgatcg
cgtctacatt 3600aacgtggtgg tgaagaataa agaatatcgt ctggcgacca
atgctagtca ggcgggcgtg 3660gagaaaattc tgtctgcact tgaaatcccg
gatgtgggta atttatccca ggtggttgtg 3720atgaaaagta aaaatgacca
agggatcacc aataaatgca aaatgaatct gcaagataac 3780aacggcaacg
acattggttt tatcggcttc caccaattca ataatatcgc gaagcttgtg
3840gcctcaaatt ggtacaaccg tcagattgag cgcagctccc gcactttagg
ctgtagctgg 3900gagttcattc cggtagatga cggttgggga gaacgcccat tgtaa
39455331314PRTArtificial SequenceBoNT/A comprising FXa (N872) and
Thrombin (E884) 533Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp
Pro Val Asn Gly1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn
Ala Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His Asn
Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro
Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val
Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn
Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100
105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu
Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly
Pro Ser Ala Asp Ile145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe
Gly His Glu Val Leu Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser
Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala
Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225
230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly
His Asp Ala Lys 260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe
Arg Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr
Leu Asn Lys Ala Lys Ser Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu
Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320 Tyr Leu Leu
Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys
Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345
350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn
355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val
Asn Tyr 370 375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn
Leu Ala Ala Asn385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn
Asn Met Asn Phe Thr Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe
Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser
Lys Gly Gly Gly Ser Gly Gly Gly Glu Asn Leu 435 440 445 Tyr Phe Gln
Gly Gly Gly Ser Gly Gly Gly Ala Leu Asn Asp Leu Cys 450 455 460 Ile
Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn465 470
475 480 Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr
Asn 485 490 495 Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile
Gln Gln Tyr 500 505 510 Tyr Leu Thr
Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu 515 520 525 Asn
Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met Pro Asn Ile 530 535
540 Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr Thr
Met545 550 555 560 Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly
Lys Ser Arg Ile 565 570 575 Ala Leu Thr Asn Ser Val Asn Glu Ala Leu
Leu Asn Pro Ser Arg Val 580 585 590 Tyr Thr Phe Phe Ser Ser Asp Tyr
Val Lys Lys Val Asn Lys Ala Thr 595 600 605 Glu Ala Ala Met Phe Leu
Gly Trp Val Glu Gln Leu Val Tyr Asp Phe 610 615 620 Thr Asp Glu Thr
Ser Glu Val Ser Thr Thr Asp Lys Ile Ala Asp Ile625 630 635 640 Thr
Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met 645 650
655 Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe Ser Gly Ala Val
660 665 670 Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu
Gly Thr 675 680 685 Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val Leu
Thr Val Gln Thr 690 695 700 Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu
Lys Trp Asp Glu Val Tyr705 710 715 720 Lys Tyr Ile Val Thr Asn Trp
Leu Ala Lys Val Asn Thr Gln Ile Asp 725 730 735 Leu Ile Arg Lys Lys
Met Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala 740 745 750 Thr Lys Ala
Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu 755 760 765 Lys
Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys Leu Asn 770 775
780 Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu Asn
Gln785 790 795 800 Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro
Tyr Gly Val Lys 805 810 815 Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys
Asp Ala Leu Leu Lys Tyr 820 825 830 Ile Tyr Asp Asn Arg Gly Thr Leu
Ile Gly Gln Val Asp Arg Leu Lys 835 840 845 Asp Lys Val Asn Asn Thr
Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser 850 855 860 Lys Tyr Val Asp
Asn Gln Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile865 870 875 880 Lys
Asn Ile Glu Gly Arg Ile Asn Thr Ser Ile Leu Asn Leu Arg Tyr 885 890
895 Glu Leu Val Pro Arg Gly Ser Asn His Leu Ile Asp Leu Ser Arg Tyr
900 905 910 Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro
Ile Asp 915 920 925 Lys Asn Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser
Lys Ile Glu Val 930 935 940 Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser
Met Tyr Glu Asn Phe Ser945 950 955 960 Thr Ser Phe Trp Ile Arg Ile
Pro Lys Tyr Phe Asn Ser Ile Ser Leu 965 970 975 Asn Asn Glu Tyr Thr
Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp 980 985 990 Lys Val Ser
Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr 995 1000 1005
Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn
1010 1015 1020 Ile Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile
Thr Asn Asn1025 1030 1035 1040Arg Leu Asn Asn Ser Lys Ile Tyr Ile
Asn Gly Arg Leu Ile Asp Gln 1045 1050 1055 Lys Pro Ile Ser Asn Leu
Gly Asn Ile His Ala Ser Asn Asn Ile Met 1060 1065 1070 Phe Lys Leu
Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys 1075 1080 1085
Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp
1090 1095 1100 Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp
Phe Trp Gly1105 1110 1115 1120Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr
Tyr Met Leu Asn Leu Tyr Asp 1125 1130 1135 Pro Asn Lys Tyr Val Asp
Val Asn Asn Val Gly Ile Arg Gly Tyr Met 1140 1145 1150 Tyr Leu Lys
Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu 1155 1160 1165
Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala
1170 1175 1180 Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg
Val Tyr Ile1185 1190 1195 1200Asn Val Val Val Lys Asn Lys Glu Tyr
Arg Leu Ala Thr Asn Ala Ser 1205 1210 1215 Gln Ala Gly Val Glu Lys
Ile Leu Ser Ala Leu Glu Ile Pro Asp Val 1220 1225 1230 Gly Asn Leu
Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly 1235 1240 1245
Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp
1250 1255 1260 Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala
Lys Leu Val1265 1270 1275 1280Ala Ser Asn Trp Tyr Asn Arg Gln Ile
Glu Arg Ser Ser Arg Thr Leu 1285 1290 1295 Gly Cys Ser Trp Glu Phe
Ile Pro Val Asp Asp Gly Trp Gly Glu Arg 1300 1305 1310 Pro Leu
5343921DNAArtificial SequenceDNA encoding a BoNT/A comprising MMP-9
(I870) 534atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt
ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa
aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc
cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg
tcttactatg atagcacata cctgagtacc 240gacaatgaaa aggacaacta
cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300acagatctcg
gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt
360agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt
gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca
ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat
gaagtcctga atctgacgcg gaatggctat 540ggatcgacgc agtatattcg
tttttctcca gatttcacat ttggatttga agaaagcctc 600gaagttgata
cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc
660ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat
taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt
cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat
gcaaaattta ttgatagtct gcaagaaaac 840gaatttcggc tgtactatta
caataaattc aaagacattg catcaacctt aaacaaggcg 900aaaagcattg
tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa
960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa
atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact
ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat
aaagccgtct ttaagatcaa catcgtaccg 1140aaagttaact acaccatcta
tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200tttaacggcc
agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc
1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag
caaaggcggt 1320ggttctggcg gtggtgaaaa cctgtacttc cagggcggtg
gctccggtgg tggtgcgctc 1380aatgatttat gcatcaaggt gaacaactgg
gacttgtttt tctctccatc tgaagataat 1440tttactaacg acttgaacaa
aggagaggaa attacttccg ataccaacat cgaagcagcg 1500gaagagaata
ttagtctaga tcttattcaa caatattacc tgacctttaa ttttgataac
1560gagcctgaga acatttccat tgagaatctc agctctgaca tcatcggcca
gctggaactg 1620atgccgaata tcgaacgctt tcctaatgga aagaaatatg
aattggacaa atacaccatg 1680ttccactatc tccgcgcgca ggagtttgag
cacggcaagt ctcgtattgc tctgaccaat 1740tcggtaaacg aagccctttt
aaatccttcg cgtgtgtaca cctttttctc aagcgattat 1800gttaaaaaag
tgaacaaggc gaccgaagcg gcgatgtttt tgggatgggt ggaacaactg
1860gtatatgact ttacggatga aacttctgaa gtctcgacca ccgacaaaat
tgccgatatt 1920accattatca ttccctatat tggccctgca ctgaacattg
gtaacatgct gtataaagat 1980gattttgtgg gcgccctgat cttttcaggc
gctgttatcc tgctggaatt tatcccggaa 2040atcgccattc cagtactcgg
tacctttgcg ctggtgtcct atatcgcaaa caaagttttg 2100actgtccaga
cgatcgacaa cgcgctcagt aaacgtaacg aaaaatggga tgaggtgtat
2160aagtatattg ttaccaactg gctcgctaaa gtaaacaccc agattgacct
gattcgcaag 2220aagatgaaag aagcgctgga aaaccaagca gaagcgacca
aagctattat caactatcaa 2280tataaccagt acacagagga agaaaagaat
aacatcaact tcaacatcga cgacttatct 2340tcaaagctga atgaatctat
taacaaagcg atgattaata ttaacaagtt cttgaaccaa 2400tgtagtgtca
gctatctgat gaactcgatg atcccttacg gtgtgaaacg tctggaagac
2460ttcgatgcaa gccttaaaga tgcccttctg aagtatattt acgataatcg
cggaactctt 2520attggccaag tggatcgctt aaaagataaa gtcaacaaca
cgctgagtac agacatccct 2580tttcagctgt ctaaatatgt ggacaatcag
cgcctgctgt ccacgtttac ggaatacatc 2640ggtccactgg gtctgtgggc
acagctgaac ttgcgttacg agagtaacca tctgattgat 2700ctgagccgtt
acgcatctaa aatcaacatc ggatccaagg tgaacttcga tcctatcgac
2760aaaaaccaga ttcaattgtt caacttagaa tcgtcaaaga ttgaagttat
cttaaaaaat 2820gcgattgtat ataattcaat gtacgaaaat ttctctacga
gcttttggat tcgtattccg 2880aaatatttca acagtatctc tttaaacaac
gagtatacta tcatcaattg tatggagaat 2940aacagcgggt ggaaagtgag
ccttaactat ggtgaaatca tctggactct gcaggacact 3000caagaaatta
aacaacgcgt ggtgtttaaa tactcacaga tgattaacat ctcggattat
3060attaatcgct ggatttttgt gacaattact aacaaccggc tgaacaacag
caaaatttac 3120attaacggtc gcctgatcga tcagaaacca atcagtaatc
tcggtaacat tcacgcatcg 3180aataatatca tgttcaaact ggatggttgt
cgcgacacgc accgttacat ttggatcaaa 3240tacttcaatt tattcgacaa
agaactcaac gaaaaggaga ttaaggatct ttatgacaat 3300cagtctaatt
cgggtattct gaaagacttt tggggtgatt accttcagta cgataaaccg
3360tattatatgt taaacttata tgatccgaat aaatacgttg acgtcaacaa
cgttggcatt 3420cgcggctata tgtatctgaa agggccgcgt ggcagcgtga
tgaccactaa catttactta 3480aactcctccc tctatcgcgg tactaaattt
attatcaaga aatatgcctc tggcaacaag 3540gacaatatcg tacgcaataa
cgatcgcgtc tacattaacg tggtggtgaa gaataaagaa 3600tatcgtctgg
cgaccaatgc tagtcaggcg ggcgtggaga aaattctgtc tgcacttgaa
3660atcccggatg tgggtaattt atcccaggtg gttgtgatga aaagtaaaaa
tgaccaaggg 3720atcaccaata aatgcaaaat gaatctgcaa gataacaacg
gcaacgacat tggttttatc 3780ggcttccacc aattcaataa tatcgcgaag
cttgtggcct caaattggta caaccgtcag 3840attgagcgca gctcccgcac
tttaggctgt agctgggagt tcattccggt agatgacggt 3900tggggagaac
gcccattgta a 39215351306PRTArtificial SequenceBoNT/A comprising
MMP-9 (I870) 535Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro
Val Asn Gly1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys
Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu
Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro
Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu
Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg
Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105
110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys
115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly
Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro
Ser Ala Asp Ile145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr
Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu
Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly
Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 Leu
Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230
235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly
Leu 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His
Asp Ala Lys 260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg
Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu
Asn Lys Ala Lys Ser Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln
Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser
Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe
Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350
Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355
360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn
Tyr 370 375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu
Ala Ala Asn385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn
Met Asn Phe Thr Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys
Gly Gly Gly Ser Gly Gly Gly Glu Asn Leu 435 440 445 Tyr Phe Gln Gly
Gly Gly Ser Gly Gly Gly Ala Leu Asn Asp Leu Cys 450 455 460 Ile Lys
Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn465 470 475
480 Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn
485 490 495 Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln
Gln Tyr 500 505 510 Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn
Ile Ser Ile Glu 515 520 525 Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu
Glu Leu Met Pro Asn Ile 530 535 540 Glu Arg Phe Pro Asn Gly Lys Lys
Tyr Glu Leu Asp Lys Tyr Thr Met545 550 555 560 Phe His Tyr Leu Arg
Ala Gln Glu Phe Glu His Gly Lys Ser Arg Ile 565 570 575 Ala Leu Thr
Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser Arg Val 580 585 590 Tyr
Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr 595 600
605 Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr Asp Phe
610 615 620 Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala
Asp Ile625 630 635 640 Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu
Asn Ile Gly Asn Met 645 650 655 Leu Tyr Lys Asp Asp Phe Val Gly Ala
Leu Ile Phe Ser Gly Ala Val 660 665 670 Ile Leu Leu Glu Phe Ile Pro
Glu Ile Ala Ile Pro Val Leu Gly Thr 675 680 685 Phe Ala Leu Val Ser
Tyr Ile Ala Asn Lys Val Leu Thr Val Gln Thr 690 695 700 Ile Asp Asn
Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu Val Tyr705 710 715 720
Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp 725
730 735 Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala Glu
Ala 740 745 750 Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr
Glu Glu Glu 755 760 765 Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu
Ser Ser Lys Leu Asn 770 775 780 Glu Ser Ile Asn Lys Ala Met Ile Asn
Ile Asn Lys Phe Leu Asn Gln785 790 795 800 Cys Ser Val Ser Tyr Leu
Met Asn Ser Met Ile Pro Tyr Gly Val Lys 805 810 815 Arg Leu Glu Asp
Phe Asp Ala Ser Leu Lys Asp Ala Leu Leu Lys Tyr 820 825 830 Ile Tyr
Asp Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg Leu Lys 835 840 845
Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser 850
855 860 Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Phe Thr Glu Tyr
Ile865 870 875 880 Gly Pro Leu Gly Leu Trp Ala Gln Leu Asn Leu
Arg Tyr Glu Ser Asn 885 890 895 His Leu Ile Asp Leu Ser Arg Tyr Ala
Ser Lys Ile Asn Ile Gly Ser 900 905 910 Lys Val Asn Phe Asp Pro Ile
Asp Lys Asn Gln Ile Gln Leu Phe Asn 915 920 925 Leu Glu Ser Ser Lys
Ile Glu Val Ile Leu Lys Asn Ala Ile Val Tyr 930 935 940 Asn Ser Met
Tyr Glu Asn Phe Ser Thr Ser Phe Trp Ile Arg Ile Pro945 950 955 960
Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn 965
970 975 Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly
Glu 980 985 990 Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys Gln
Arg Val Val 995 1000 1005 Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser
Asp Tyr Ile Asn Arg Trp 1010 1015 1020 Ile Phe Val Thr Ile Thr Asn
Asn Arg Leu Asn Asn Ser Lys Ile Tyr1025 1030 1035 1040Ile Asn Gly
Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn Leu Gly Asn 1045 1050 1055
Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly Cys Arg Asp
1060 1065 1070 Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe
Asp Lys Glu 1075 1080 1085 Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr
Asp Asn Gln Ser Asn Ser 1090 1095 1100 Gly Ile Leu Lys Asp Phe Trp
Gly Asp Tyr Leu Gln Tyr Asp Lys Pro1105 1110 1115 1120Tyr Tyr Met
Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn 1125 1130 1135
Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser
1140 1145 1150 Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr
Arg Gly Thr 1155 1160 1165 Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly
Asn Lys Asp Asn Ile Val 1170 1175 1180 Arg Asn Asn Asp Arg Val Tyr
Ile Asn Val Val Val Lys Asn Lys Glu1185 1190 1195 1200Tyr Arg Leu
Ala Thr Asn Ala Ser Gln Ala Gly Val Glu Lys Ile Leu 1205 1210 1215
Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln Val Val Val
1220 1225 1230 Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys Cys
Lys Met Asn 1235 1240 1245 Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly
Phe Ile Gly Phe His Gln 1250 1255 1260 Phe Asn Asn Ile Ala Lys Leu
Val Ala Ser Asn Trp Tyr Asn Arg Gln1265 1270 1275 1280Ile Glu Arg
Ser Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro 1285 1290 1295
Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1300 1305
5363916DNAArtificial SequenceDNA encoding a BoNT/A comprising Fxa
(N872) 536atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt
ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa
aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc
cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg
tcttactatg atagcacata cctgagtacc 240gacaatgaaa aggacaacta
cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300acagatctcg
gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt
360agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt
gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca
ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat
gaagtcctga atctgacgcg gaatggctat 540ggatcgacgc agtatattcg
tttttctcca gatttcacat ttggatttga agaaagcctc 600gaagttgata
cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc
660ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat
taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt
cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat
gcaaaattta ttgatagtct gcaagaaaac 840gaatttcggc tgtactatta
caataaattc aaagacattg catcaacctt aaacaaggcg 900aaaagcattg
tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa
960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa
atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact
ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat
aaagccgtct ttaagatcaa catcgtaccg 1140aaagttaact acaccatcta
tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200tttaacggcc
agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc
1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag
caaaggcggt 1320ggttctggcg gtggtgaaaa cctgtacttc cagggcggtg
gctccggtgg tggtgcgctc 1380aatgatttat gcatcaaggt gaacaactgg
gacttgtttt tctctccatc tgaagataat 1440tttactaacg acttgaacaa
aggagaggaa attacttccg ataccaacat cgaagcagcg 1500gaagagaata
ttagtctaga tcttattcaa caatattacc tgacctttaa ttttgataac
1560gagcctgaga acatttccat tgagaatctc agctctgaca tcatcggcca
gctggaactg 1620atgccgaata tcgaacgctt tcctaatgga aagaaatatg
aattggacaa atacaccatg 1680ttccactatc tccgcgcgca ggagtttgag
cacggcaagt ctcgtattgc tctgaccaat 1740tcggtaaacg aagccctttt
aaatccttcg cgtgtgtaca cctttttctc aagcgattat 1800gttaaaaaag
tgaacaaggc gaccgaagcg gcgatgtttt tgggatgggt ggaacaactg
1860gtatatgact ttacggatga aacttctgaa gtctcgacca ccgacaaaat
tgccgatatt 1920accattatca ttccctatat tggccctgca ctgaacattg
gtaacatgct gtataaagat 1980gattttgtgg gcgccctgat cttttcaggc
gctgttatcc tgctggaatt tatcccggaa 2040atcgccattc cagtactcgg
tacctttgcg ctggtgtcct atatcgcaaa caaagttttg 2100actgtccaga
cgatcgacaa cgcgctcagt aaacgtaacg aaaaatggga tgaggtgtat
2160aagtatattg ttaccaactg gctcgctaaa gtaaacaccc agattgacct
gattcgcaag 2220aagatgaaag aagcgctgga aaaccaagca gaagcgacca
aagctattat caactatcaa 2280tataaccagt acacagagga agaaaagaat
aacatcaact tcaacatcga cgacttatct 2340tcaaagctga atgaatctat
taacaaagcg atgattaata ttaacaagtt cttgaaccaa 2400tgtagtgtca
gctatctgat gaactcgatg atcccttacg gtgtgaaacg tctggaagac
2460ttcgatgcaa gccttaaaga tgcccttctg aagtatattt acgataatcg
cggaactctt 2520attggccaag tggatcgctt aaaagataaa gtcaacaaca
cgctgagtac agacatccct 2580tttcagctgt ctaaatatgt ggacaatcag
cgcctgctgt ccacgtttac ggaatacatc 2640aaaaacattg agggccgtat
caacactagt attctgatct aaccatctga ttgatctgag 2700ccgttacgca
tctaaaatca acatcggatc caaggtgaac ttcgatccta tcgacaaaaa
2760ccagattcaa ttgttcaact tagaatcgtc aaagattgaa gttatcttaa
aaaatgcgat 2820tgtatataat tcaatgtacg aaaatttctc tacgagcttt
tggattcgta ttccgaaata 2880tttcaacagt atctctttaa acaacgagta
tactatcatc aattgtatgg agaataacag 2940cgggtggaaa gtgagcctta
actatggtga aatcatctgg actctgcagg acactcaaga 3000aattaaacaa
cgcgtggtgt ttaaatactc acagatgatt aacatctcgg attatattaa
3060tcgctggatt tttgtgacaa ttactaacaa ccggctgaac aacagcaaaa
tttacattaa 3120cggtcgcctg atcgatcaga aaccaatcag taatctcggt
aacattcacg catcgaataa 3180tatcatgttc aaactggatg gttgtcgcga
cacgcaccgt tacatttgga tcaaatactt 3240caatttattc gacaaagaac
tcaacgaaaa ggagattaag gatctttatg acaatcagtc 3300taattcgggt
attctgaaag acttttgggg tgattacctt cagtacgata aaccgtatta
3360tatgttaaac ttatatgatc cgaataaata cgttgacgtc aacaacgttg
gcattcgcgg 3420ctatatgtat ctgaaagggc cgcgtggcag cgtgatgacc
actaacattt acttaaactc 3480ctccctctat cgcggtacta aatttattat
caagaaatat gcctctggca acaaggacaa 3540tatcgtacgc aataacgatc
gcgtctacat taacgtggtg gtgaagaata aagaatatcg 3600tctggcgacc
aatgctagtc aggcgggcgt ggagaaaatt ctgtctgcac ttgaaatccc
3660ggatgtgggt aatttatccc aggtggttgt gatgaaaagt aaaaatgacc
aagggatcac 3720caataaatgc aaaatgaatc tgcaagataa caacggcaac
gacattggtt ttatcggctt 3780ccaccaattc aataatatcg cgaagcttgt
ggcctcaaat tggtacaacc gtcagattga 3840gcgcagctcc cgcactttag
gctgtagctg ggagttcatt ccggtagatg acggttgggg 3900agaacgccca ttgtaa
39165371309PRTArtificial SequenceBoNT/A comprising Fxa (N872)
537Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1
5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln
Pro 20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile
Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu
Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr
Asp Ser Thr Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr
Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr
Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile
Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val
Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135
140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp
Ile145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val
Leu Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile
Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu
Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala
Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala
Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240 Arg
Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250
255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys
260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr
Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala
Lys Ser Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys
Asn Val Phe Lys Glu Lys305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr
Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu
Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val
Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe
Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375
380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala
Asn385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn
Phe Thr Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr
Lys Leu Leu Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys Gly Gly
Gly Ser Gly Gly Gly Glu Asn Leu 435 440 445 Tyr Phe Gln Gly Gly Gly
Ser Gly Gly Gly Ala Leu Asn Asp Leu Cys 450 455 460 Ile Lys Val Asn
Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn465 470 475 480 Phe
Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn 485 490
495 Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln Gln Tyr
500 505 510 Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser
Ile Glu 515 520 525 Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu
Met Pro Asn Ile 530 535 540 Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
Leu Asp Lys Tyr Thr Met545 550 555 560 Phe His Tyr Leu Arg Ala Gln
Glu Phe Glu His Gly Lys Ser Arg Ile 565 570 575 Ala Leu Thr Asn Ser
Val Asn Glu Ala Leu Leu Asn Pro Ser Arg Val 580 585 590 Tyr Thr Phe
Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr 595 600 605 Glu
Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr Asp Phe 610 615
620 Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala Asp
Ile625 630 635 640 Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn
Ile Gly Asn Met 645 650 655 Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
Ile Phe Ser Gly Ala Val 660 665 670 Ile Leu Leu Glu Phe Ile Pro Glu
Ile Ala Ile Pro Val Leu Gly Thr 675 680 685 Phe Ala Leu Val Ser Tyr
Ile Ala Asn Lys Val Leu Thr Val Gln Thr 690 695 700 Ile Asp Asn Ala
Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu Val Tyr705 710 715 720 Lys
Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp 725 730
735 Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala
740 745 750 Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu
Glu Glu 755 760 765 Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser
Ser Lys Leu Asn 770 775 780 Glu Ser Ile Asn Lys Ala Met Ile Asn Ile
Asn Lys Phe Leu Asn Gln785 790 795 800 Cys Ser Val Ser Tyr Leu Met
Asn Ser Met Ile Pro Tyr Gly Val Lys 805 810 815 Arg Leu Glu Asp Phe
Asp Ala Ser Leu Lys Asp Ala Leu Leu Lys Tyr 820 825 830 Ile Tyr Asp
Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg Leu Lys 835 840 845 Asp
Lys Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser 850 855
860 Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Phe Thr Glu Tyr
Ile865 870 875 880 Lys Asn Ile Glu Gly Arg Ile Asn Thr Ser Ile Leu
Asn Leu Arg Tyr 885 890 895 Glu Ser Asn His Leu Ile Asp Leu Ser Arg
Tyr Ala Ser Lys Ile Asn 900 905 910 Ile Gly Ser Lys Val Asn Phe Asp
Pro Ile Asp Lys Asn Gln Ile Gln 915 920 925 Leu Phe Asn Leu Glu Ser
Ser Lys Ile Glu Val Ile Leu Lys Asn Ala 930 935 940 Ile Val Tyr Asn
Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp Ile945 950 955 960 Arg
Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr Thr 965 970
975 Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn
980 985 990 Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile
Lys Gln 995 1000 1005 Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn
Ile Ser Asp Tyr Ile 1010 1015 1020 Asn Arg Trp Ile Phe Val Thr Ile
Thr Asn Asn Arg Leu Asn Asn Ser1025 1030 1035 1040Lys Ile Tyr Ile
Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn 1045 1050 1055 Leu
Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly 1060
1065 1070 Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn
Leu Phe 1075 1080 1085 Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp
Leu Tyr Asp Asn Gln 1090 1095 1100 Ser Asn Ser Gly Ile Leu Lys Asp
Phe Trp Gly Asp Tyr Leu Gln Tyr1105 1110 1115 1120Asp Lys Pro Tyr
Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val 1125 1130 1135 Asp
Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro 1140
1145 1150 Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser
Leu Tyr 1155 1160 1165 Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala
Ser Gly Asn Lys Asp 1170 1175 1180 Asn Ile Val Arg Asn Asn Asp Arg
Val Tyr Ile Asn Val Val Val Lys1185 1190 1195 1200Asn Lys Glu Tyr
Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu 1205 1210 1215 Lys
Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln 1220
1225 1230 Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn
Lys Cys 1235 1240 1245 Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp
Ile Gly Phe Ile Gly 1250 1255 1260 Phe His Gln Phe Asn Asn
Ile Ala Lys Leu Val Ala Ser Asn Trp Tyr1265 1270 1275 1280Asn Arg
Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp Glu 1285 1290
1295 Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1300
1305
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